Method of using reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins

The present invention discloses eight new reduced dimensionality (RD) triple resonance nuclear magnetic resonance (NMR) experiments for measuring chemical shift values of certain nuclei in a protein molecule. The RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D H,C,(C-TOCSY—CO),N,HN NMR experiment are designed to yield “sequential” connectivities, while the RD 3D Hαβ,Cαβ,CO,HA NMR experiment and the RD 3D Hαβ,Cαβ,N,HN NMR experiment provide “intraresidue” connectivities. The RD 3D H,C,C,H—COSY NMR experiment, the RD 3D H,C,C,H-TOCSY NMR experiment, and the RD 2D H,C,H—COSY NMR experiment allow one to obtain assignments for aliphatic and aromatic side chain chemical shifts, while the RD 2D HB,CB,(CG,CD),HD NMR experiment provide information for the aromatic side chain chemical shifts. In addition, a method of conducting suites of RD triple resonance NMR experiments for high-throughput resonance assignment of proteins and identification of the location of secondary structure elements are disclosed.

FIELD OF THE INVENTION

The present invention relates to methods of using reduced dimensionality nuclear magnetic resonance (NMR) spectroscopy for obtaining chemical shift assignment and structure determination of proteins.

BACKGROUND OF THE INVENTION

The use of triple resonance (TR) nuclear magnetic resonance (NMR) experiments for the resonance assignment of polypeptide chains via heteronuclear scalar connectivities (Montelione et al.,J. Am Chem. Soc.,111:5474–5475 (1989); Montelione et al.,J. Magn. Reson.,87:183–188 (1989); Kay et al.,J. Magn. Reson.,89:496–514 (1990); Ikura et al.,Biochemistry,29:4659–8979 (1990); Edison et al.,Methods Enzymol.,239:3–79 (1994)) is a standard approach which neatly complements the assignment protocol based on1H—1H nuclear Overhauser effects (NOE) (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986)). In addition, triple resonance NMR spectra are highly amenable to a fast automated analysis (Friedrichs et al.,J. Biomol. NMR,4:703–726 (1994); Zimmerman et al.,J. Biomol. NMR,4:241–256 (1994); Bartels et al.,J. Biomol. NMR,7:207–213 (1996); Morelle et al.,J. Biomol. NMR,5:154–160 (1995); Buchler et al.,J. Magn. Reson.,125:34–42 (1997); Lukin et al.,J. Biomol. NMR,9:151–166 (1997)), yielding the13Cα/βchemical shifts at an early stage of the assignment procedure. This enables both, the identification of regular secondary structure elements without reference to NOEs (Spera et al.,J. Am. Chem. Soc.,113: 5490–5491 (1991)) and the derivation of (φ,ψ)-angle constraints which serve to reduce the number of cycles consisting of nuclear Overhauser enhancement spectroscopy (NOESY) peak assignment and structure calculation (Luginbühl et al.,J. Magn. Reson., B 109:229–233 (1995)).

NMR assignments are prerequisite for NMR-based structural biology (Wüthrich,NMR of Proteins and Nucleic Acid, Wiley:New York (1986)) and, thus, for high-throughput (HTP) structure determination in structural genomics (Rost,Structure,6:259–263 (1998); Montelione et al.,Nature Struct. Biol.,6:11–12. (1999); Burley,Nature Struc Biol.,7:932–934 (2000)) and for exploring structure-activity relationships (SAR) by NMR for drug discovery (Shuker et al.,Science,274:1531–1534 (1996)). The aims of structural genomics are to (i) explore the naturally occurring “protein fold space” and (ii) contribute to the characterization of function through the assignment of atomic resolution three-dimensional (3D) structures to proteins. It is now generally acknowledged that NMR will play an important role in structural genomics (Montelione et al.,Nature Struc. Biol.,7:982–984 (2000)). The resulting demand for HTP structure determination requires fast and automated NMR data collection and analysis protocols (Moseley et al.,Curr. Opin. Struct. Biol.,9:635–642 (1999)).

The establishment of a HTP NMR structural genomics pipeline requires two key objectives in data collection. Firstly, the measurement time should be minimized in order to (i) lower the cost per structure and (ii) relax the constraint that NMR samples need to be stable over a long period of measurement time. The recent introduction of commercial cryogenic probes (Styles et al.,J. Magn. Reson.,60:397–404 (1984); Flynn et al.,J. Am Chem. Soc.,122:4823–4824 (2000)) promises to reduce measurement times by about a factor of ten or more, and will greatly impact the realization of this first objective. Secondly, reliable automated spectral analysis requires recording of a “redundant” set of multidimensional NMR experiments each affording good resolution (which requires appropriately long maximal evolution times in all indirect dimensions). Concomitantly, it is desirable to keep the total number of NMR spectra small in order to minimize “interspectral” variations of chemical shift measurements, which may impede automated spectral analysis. Straightforward consideration of this second objective would suggest increasing the dimensionality of the spectra, preferably by implementing a suite of four- or even higher-dimensional NMR experiments. Importantly, however, the joint realization of the first and second objectives is tightly limited by the rather large lower bounds of higher-dimensional TR NMR measurement times if appropriately long maximal evolution times are chosen.

Hence, “sampling limited” and “sensitivity limited” data collection regimes are distinguished, depending on whether the sampling of the indirect dimensions or the sensitivity of the multidimensional NMR experiments “per se” determines the minimally achievable measurement time. As a matter of fact, the ever increasing performance of NMR spectrometers will soon lead to the situation where, for many protein samples, the sensitivity of the NMR spectrometers do not constitute the prime bottleneck determining minimal measurement times. Instead, the minimal measurement times encountered for recording conventional higher-dimensional NMR schemes will be “sampling limited,” particularly as high sensitivity cryoprobes become generally available. As structure determinations of proteins rely on nearly complete assignment of chemical shifts, which are obtained using multidimensional13C,15N,1H-TR NMR experiments (Montelione et al.,J. Am Chem. Soc.,111:5474–5475 (1989); Montelione, et al.,J. Magn. Reson.,87:183–188 (1989); Ikura et al.,Biochemistry,29:4659–8979 (1990)), the development of TR NMR techniques that avoid the sampling limited regime represents a key challenge for future biomolecular NMR methods development.

The present invention is directed to overcoming the deficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a method of conducting a reduced dimensionality three-dimensional (3D)HA,CA,(CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) an α-proton of amino acid residue i−1,1Hαi−1; (2) an α-carbon of amino acid residue i−1,13Cαi−1; (3) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (4) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαi−1and13Cαi−1of amino acid residue i−1 are connected to the chemical shift evolutions of15Niand1HNiof amino acid residue i, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi−1and15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα) and t2(15N), respectively, and the chemical shift value of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of1Hαi−1. Then, the NMR signals are processed to generate a 3D NMR spectrum with a primary peak pair derived from the cosine modulating, where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) the chemical shift values of1Hαi−1and13Cαi−1are measured in a frequency domain dimension, ω1(13Cα), by the frequency difference between the two peaks forming the primary peak pair and the frequency at the center of the two peaks, respectively.

The present invention also relates to a method of conducting a reduced dimensionality three-dimensional (3D)H,C,(C-TOCSY—CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) aliphatic protons of amino acid residue i−1,1Halii−1; (2) aliphatic carbons of amino acid residue i−1,13Calii−1; (3) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (4) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Halii−1and13Calii−1of amino acid residue i−1 are connected to the chemical shift evolutions of15Niand1HNiof amino acid residue i, under conditions effective (1) to generate a NMR signal encoding the chemical shifts of13Calii−1and15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cali) and t2(15N), respectively, and the chemical shift of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the chemical shift evolutions of13Calii−1in t1(13Cali) with the chemical shift evolutions of1Halii−1. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) the chemical shift values of1Halii−1and13Calii−1are measured in a frequency domain dimension, ω1(13Cali), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

Another aspect of the present invention relates to a method of conducting a reduced dimensionality three-dimensional (3D)Hα/β,Cα/β,CO,HA nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,1Hβi; (2) a β-carbon of amino acid residue i,13Cβi; (3) an α-proton of amino acid residue i,1Hαi; (4) an α-carbon of amino acid residue i,13Cαi; and (5) a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαl,1Hβl,13Cαl, and13Cβiare connected to the chemical shift evolution of13C′i, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi,13Cβiand13C′iin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα/β) and t2(13C′), respectively, and the chemical shift value of1Hαiin a direct time domain dimension, t3(1Hα), and (2) to cosine modulate the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβi, respectively. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13C′iand1Hαiare measured in two frequency domain dimensions, ω2(13C′) and ω3(1Hα), respectively, and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between the two peaks forming the peak pairs, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

A further aspect of the present invention relates to a method of conducting a reduced dimensionality three-dimensional (3D)Hα/β,Cα/β,N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,1Hβi; (2) a β-carbon of amino acid residue i,13Cβi; (3) an α-proton of amino acid residue i,1Hαi; (4) an α-carbon of amino acid residue i,13Cαi; (5) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (6) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαi,1Hβi,13Cαi, and13Cβiare connected to the chemical shift evolutions of15Niand1HNi, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi,13Cβiand15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα/β) and t2(15N), respectively, and the chemical shift value of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβi, respectively. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between the two peaks forming the peak pairs, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

The present invention also relates to a method of conducting a reduced dimensionality three-dimensional (3D)H,C,C,H—COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm,1Hn, and13Cnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups that are linked by a single covalent carbon—carbon bond in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effects a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolutions of1Hnand13Cn, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in two indirect time domain dimensions, t1(13Cm) and t2(13Cn), respectively, and the chemical shift value of1Hnin a direct time domain dimension, t3(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13Cnand1Hnare measured in two frequency domain dimensions, ω2(13Cn) and ω3(1Hn), respectively, and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

Another aspect of the present invention relates to a method of conducting a reduced dimensionality three-dimensional (3D)H,C,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm,1Hn, and13Cnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups that may or may not be directly linked by a single covalent carbon-carbon bond in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolutions of1Hnand13Cn, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in two indirect time domain dimensions, t1(13Cm) and t2(13Cn), and the chemical shift value of1Hnin a direct time domain dimension, t3(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13Cnand1Hnare measured in two frequency domain dimensions, ω2(13Cn) and ω3(1Hn), respectively, and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

A further aspect of the present invention relates to a method of conducting a reduced dimensionality two-dimensional (2D)HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule: (1) a β-proton of an amino acid residue with an aromatic side chain,1Hβ; (2) a β-carbon of an amino acid residue with an aromatic side chain,13Cβ; and (3) a δ-proton of an amino acid residue with an aromatic side chain,1Hδ. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hβand13Cβare connected to the chemical shift evolution of1Hδ, under conditions effective (1) to generate NMR signals encoding the chemical shift value of13Cβin a phase sensitive manner in an indirect time domain dimension, t1(13Cβ), and the chemical shift value of1Hδin a direct time domain dimension, t2(1Hδ), and (2) to cosine modulate the chemical shift evolution of13Cβin t1(13Cβ) with the chemical shift evolution of1Hβ. Then, the NMR signals are processed to generate a 2D NMR spectrum with a peak pair derived from the cosine modulating where (1) the chemical shift value of1Hδis measured in a frequency domain dimension, ω2(1Hδ), and (2) the chemical shift values of1Hβand13Cβare measured in a frequency domain dimension, ω1(13Cβ), by the frequency difference between the two peaks forming the peak pair and the frequency at the center of the two peaks, respectively.

The present invention also relates to a method of conducting a reduced dimensionality two-dimensional (2D)H,C,H—COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm, and1Hnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolution of1Hn, under conditions effective (1) to generate NMR signals encoding the chemical shift value of13Cmin a phase sensitive manner in an indirect time domain dimension, t1(13Cm), and the chemical shift value of1Hnin a direct time domain dimension, t2(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 2D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift value of1Hnis measured in a frequency domain dimension, ω2(1Hn), and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

Another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton,1Hα, an α-carbon,13Cα, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD three-dimensional (3D)HA,CA,(CO),N,HN NMR experiment to measure and connect chemical shift values of the α-proton of amino acid residue i−1,1Hαi−1, the α-carbon of amino acid residue i−1,13Cαi−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3D HNNCAHANMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hα,13Cα,15N, and1HNare obtained by (i) matching the chemical shift values of1Hαi−1and13Cαi−1with the chemical shift values of1Hαiand13Cαi, (ii) using the chemical shift values of1Hαi−1and13Cαi−1to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain.

Yet another aspect of the present invention relates to a method for sequentially assigning chemical shift values of a β-proton,1Hβ, a β-carbon,13Cβ, an α-proton,1Hα, an α-carbon,13Cα, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DHα/βCα/β(CO)NHN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i−1,1Hβi−1, the β-carbon of amino acid residue i−1,13Cβi−1, the α-proton of amino acid residue i−1,1Hαi−1, the α-carbon of amino acid residue i−1,13Cβi−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,iHβi, the β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNare obtained by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,1Hα/βi−1, and the α- and β-carbons of amino acid residue i−1,13Cα/βi−1, with the chemical shift values of1Hα/βiand13Cα/βi, (ii) using the chemical shift values of1Hα/βi−1and13Cα/βi−1to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain.

A further aspect of the present invention involves a method for sequentially assigning chemical shift values of aliphatic protons,1Hali, aliphatic carbons,13Cali, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,1Halii−1, the aliphatic carbons of amino acid residue i−1,13Calii−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,1Hβi, the β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hali,13Cali,15N, and1HNare obtained by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,1Hα/βi−1, and the α- and β-carbons of amino acid residue i−1,13Cα/βi−1, with the chemical shift values of1Hα/βiand13Cα/βiof amino acid residue i, (ii) using the chemical shift values of1Halii−1and13Calii−1to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain.

The present invention also relates to a method for sequentially assigning chemical shift values of aliphatic protons,1Hali, aliphatic carbons,13Cali, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,1Halii−1, the aliphatic carbons of amino acid residue i−1,13Calii−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3D HNNCAHANMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hali,13Cali,15N, and1HNare obtained by (i) matching the chemical shift values of the α-proton of amino acid residue i−1,1Hαi−1, and the α-carbon of amino acid residue i−1,13Cαi−1, with the chemical shift values of1Hαiand13Cαi, (ii) using the chemical shift values of1Halii−1and13Calii−1to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain.

Another aspect of the present invention involves a method for obtaining assignments of chemical shift values of1H,13C and15N of a protein molecule. The method involves providing a protein sample and conducting four reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, where (1) a first experiment is selected from the group consisting of a RD 3DHα/βCα/β(CO)NHN NMR experiment, a RD 3DHA,CA,(CO),N,HN NMR experiment, and a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment for obtaining sequential correlations of chemical shift values; (2) a second experiment is selected from the group consisting of a RD 3D HNNCAHANMR experiment, a RD 3DHα/β,Cα/β,N,HN NMR experiment, and a RD 3D HNN<CO,CA> NMR experiment for obtaining intraresidue correlations of chemical shift values; (3) a third experiment is a RD 3DH,C,C,H—COSY NMR experiment for obtaining assignments of sidechain chemical shift values; and (4) a fourth experiment is a RD 2DHB,CB,(CG,CD),HD NMR experiment for obtaining assignments of aromatic sidechain chemical shift values.

The present invention discloses eight new RD TR NMR experiments and different combinations of those eight experiments as well as three other RD TR NMR experiments which allows one to obtain sequential backbone chemical shift assignments for determining the secondary structure of a protein molecule and nearly complete assignments of chemical shift values for a protein molecule including aliphatic and aromatic sidechain spin systems.

RD NMR spectroscopy is a powerful approach to avoid recording TR NMR data for resonance assignment in the “sampling limited data acquisition regime.” The set of NMR experiments for HTP structure determination as claimed in the present invention allows one to effectively adapt measurement times to sensitivity requirements. This is of outstanding value in view of HTP protein resonance assignment efforts in the forthcoming era of commercially available cryogenic probes. In particular, the rapid determination of a protein's secondary structure can greatly support fold prediction and thus protein target selection required for structural genomics (Montelione et al.,Nature Struc. Biol.,7:982–984 (2000), which is hereby incorporated by reference in its entirety).

In addition, the present invention which discloses the sensitivity analysis of a suite of TR NMR experiments providing nearly complete assignments of chemical shift values of1H,13C and15N of a protein molecule is unique and, thus, of general interest for the application of TR NMR schemes. The key insights obtained from this analysis are (i) that the sensitivity of the individual NMR experiments constituting the standard set derived here is comparable or better than the 3D HNNCACB NMR experiment, which has so far been routinely employed for proteins up to about 35 kDa, (Mer et al.,J. Biomol. NMR,17:179–180 (2000), which is hereby incorporated by reference in its entirety) and (ii) that data acquisition for most samples of proteins below 20 kDa will be in the undesired sampling limited regime when using conventional NMR schemes and cryogenic probes. (For 800 MHz systems, such probes today already offer a sensitivity of 6200:1 for a standard 0.1% ethylbenzene sample (Anderson, “High Q Normal Metal NMR Probe Coils,” 42nd Experimental NMR Conference, Orlando, Fla. (2001), which is hereby incorporated by reference in its entirety).) Moreover, the sweep widths of all indirect dimensions of a multidimensional NMR experiment increase with increasing magnetic field strength (which implies increasing minimal measurement times). Hence, in view of this concomitant increase of sensitivity and sweep widths at highest magnetic fields and particularly considering the anticipated widespread use of cryogenic probes, a “change in paradigm” in biological NMR spectroscopy is expected with a new focus on research addressing the caveat of sampling limitation. This will foreseeably include development and application of data processing protocols that allow one to reduce the number of data points in the indirect dimensions without concomitantly sacrificing spectral resolution, i.e., linear prediction and maximum entropy methods (Stephenson,Prog. NMR Spectrosc.,20:515–626 (1988), which is hereby incorporated by reference in its entirety), approaches for non-linear sampling (Schmieder et al.,J. Biomol. NMR,4:483–490 (1994); Hoch, et al.,NMR Data Processing, Wiley-Liss:New York, (1996), which are hereby incorporated by reference in their entirety), and the recently introduced filter diagonalization method (Wall et al.,J. Chem. Phys.,102:8011–8022 (1995); Wall et al.,Chem. Phys. Lett.,291:465–470 (1998); Hu et al.,J. Magn. Reson.,134: 76–87 (1998), which are hereby incorporated by reference in their entirety).

Considering also random fractional deuteration of proteins for sensitivity enhancement, it is envisioned that the majority of protein structure determinations can possibly by accelerated by the application of RD NMR spectroscopy. In 2000, there were about eighty 750/800 MHz and three-hundred 600 MHz spectrometers in operation worldwide, which represent a commercial value of about $350 million (Cross,High Field NMR: a baseline study., National High Magnetic Field Laboratory, Tallahassee, Fla. (2000), which is hereby incorporated by reference in its entirety). Assuming that about 50% of the instrument time is used for NMR structure determination, it is anticipated that the application of RD NMR technology promises to greatly impact on the optimized use of the large capital invested for NMR-based structural biology.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses eight new RD TR NMR experiments and different combinations of those eight experiments as well as three other RD TR NMR experiments which allows one to obtain sequential backbone chemical shift assignments for determining the secondary structure of a protein molecule and nearly complete assignments of chemical shift values for a protein molecule including aliphatic and aromatic sidechain spin systems.FIG. 1provides a survey of (i) the names, (ii) the magnetization transfer pathways and (iii) the peak patterns observed in the projected dimension of specific embodiments of the 8 new RD NMR experiments disclosed by the present invention as well as 3 other RD NMR experiments that have previously been published. The group comprising the first three experiments are designed to yield “sequential” connectivities via one-bond scalar couplings: 3DHα/βCα/β(CO)NHN (FIG. 1A; Szyperski et al.,J. Magn. Reson., B 105: 188–191 (1994), which is hereby incorporated by reference in its entirety), 3DHACA(CO)NHN (FIG. 1B), and 3D HC(C-TOCSY—CO)NHN (FIG. 1C). The following three experiments provide “intraresidual” connectivities via one-bond scalar couplings: 3D HNNCAHA(FIG. 1D; Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety), 3DHα/βCα/βCOHA (FIG. 1E), and 3DHα/βCα/βNHN (FIG. 1F). 3D HNN<CO,CA> (FIG. 1G; Szyperski et al.,J. Magn. Reson., B 108: 197–203 (1995); Szyperski et al.,J. Am. Chem. Soc.,118:8146–8147 (1996), which are hereby incorporated by reference in their entirety) offers both intraresidual1HN—13Cαand sequential1HN—13C′ connectivities. Although 3D HNNCAHA(FIG. 1D), 3DHα/βCα/βNHN (FIG. 1F) and 3D HNN<CO,CA> (FIG. 1G) also provide sequential connectivities via two-bond13Cαi−1—15Niscalar couplings, those are usually smaller than the one-bond couplings (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety), and obtaining complete backbone resonance assignments critically depends on experiments designed to provide sequential connectivities via one-bond couplings (FIGS. 1D–F). 3DHCCH—COSY (FIG. 1H) and 3DHCCH-TOCSY (FIG. 1I) allow one to obtain assignments for the “aliphatic” side chain spin systems, while 2DHBCB(CDCG)HD (FIG. 1J) and 2D1H-TOCSY-relayedHCH—COSY (FIG. 1K) provide the corresponding information for the “aromatic” spin systems.

The RD NMR experiments are grouped accordingly in Table 1, which lists for each experiment (i) the nuclei for which the chemical shifts are measured, (ii) if and how the central peaks are acquired and (iii) additional notable technical features. State-of-the art implementations (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996); Kay,J. Am. Chem. Soc.,115:2055–2057 (1993); Grzesiek et al.,J. Magn. Reson.,99:201–207 (1992); Montelione et al.,J. Am. Chem. Soc.,114:10974–10975 (1992); Boucher et al.,J. Biomol. NMR,2:631–637 (1992); Yamazaki et al.,J. Am. Chem. Soc.,115:11054–11055 (1993); Zerbe et al.,J. Biomol. NMR,7:99–106 (1996); Grzesiek et al.,J. Biomol. NMR,3:185–204 (1993), which are hereby incorporated by reference in their entirety) making use of pulsed field z-gradients for coherence selection and/or rejection, and sensitivity enhancement (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety) were chosen, which allow executing these experiments with a single transient per acquired free induction decay (FID). Semi (Grzesiek et al.,J. Biomol. NMR,3:185–204 (1993), which is hereby incorporated by reference in its entirety) constant-time (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety) chemical shift frequency-labeling modules were used throughout in the indirect dimensions in order to minimize losses arising from transverse nuclear spin relaxation.FIGS. 2A–2Kprovide comprehensive descriptions of the RD NMR r.f. pulse sequences used in the 11 RD NMR experiments including eight previously unpublished RD NMR r.f. pulse schemes.

TABLE 1Reduced Dimensionality NMR Experiments for HTP Resonance AssignmentExperimentNuclei for which the chemical shifts areAcquisition of(see FIG. 1)correlateda,bcentral peaksc3D spectra for sequential backbone connectivities:(A)Hα/βCα/β(CO)NHN1Hβi−113Cβi−1,1Hαi−1,13Cαi−1,15Ni,1HNi13C(B)HACA(CO)NHN1Hαi−1,13Cαi−1,15Ni,1HNi13C(C)HC(C-TOCSY- CO)NHN1Ha1ii−1,13Ca1ii−1,15Ni,1HNi13C3D spectra for intraresidual backbone connectivities:(D) HNNCAHAb,d1Hαi,13Cαi,15Ni,1HNiINEPT(E)Hα/βCα/βCOHA1Hβi,13Cβi,1Hαi,13Cαi,13C═Oi13C(F)Hα/βCα/βNH1Hβi,13Cβi,1Hαi,13Cαi,15Ni,1HNi13C3D spectrum for intra- and sequential backbone connectivities:(G) HNN<CO,CA>b13C═Oi−1,13Cαi,15Ni,INEPT1HNi3D spectra for assignment of aliphatic resonances:e(H)HCCH-COSY1Hm,13Cm,1Hn,13Cn13C(I)HCCH-TOCSY1Hm,13Cm,1Hn,13Cn,1Hp,13Cp13C2D spectra for assignment of aromatic resonances:e(J)HBCB(CGCD)HD1Hβ,13Cβ,1Hδ13C(K)1H-TOCSY-HCH-COSY1Hm,13Cm,1Hn,nonefai−1, i: numbers of two sequentially located amino acid residues.bSequential connectivities via two-bond13Cαi−1-15N, scalar couplings are not considered in this table.capproach-1 (Szyperski et al.,J. Am. Chem. Soc., 118:8146–8147 (1996), which is hereby incorporated by reference in its entirety): use of incomplete INEPT (rows labeled with “INEPT”); approach-2 (Szyperski et al.,J. Am. Chem. Soc., 118:8146–8147 (1996), which is hereby incorporated by reference in its entirety): use of13C steady state magnetization (rows labeled with “13C”).dadiabatic13Cβ-decoupling (Kupce et al.,J. Magn. Reson., A 115:273–277 (1995); Matsuo et al.,J. Magn. Reson.B 113:190–194 (1996), which are hereby incorporated by reference in their entirety) is employed during delays with transverse13Cαmagnetization.em, n, p: atom numbers in neighboring CH, CH2or CH3groups.facquisition of central peaks is prevented by the use of spin-lock purge pulses (flanking the total correlation relay) to obtain pure phases.
The 3D HA,CA,(CO),N,HN Experiment

The present invention relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)HA,CA,(CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) an α-proton of amino acid residue i−1,1Hαi−1; (2) an α-carbon of amino acid residue i−1,13Cαi−1; (3) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (4) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαi−1and13Cαi−1of amino acid residue i−1 are connected to the chemical shift evolutions of15Niand1HNiof amino acid residue i, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi−1and15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα) and t2(15N), respectively, and the chemical shift value of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of1Hαi−1. Then, the NMR signals are processed to generate a 3D NMR spectrum with a primary peak pair derived from the cosine modulating, where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) the chemical shift values of1Hαi−1and13Cαi−1are measured in a frequency domain dimension, ω1(13Cα), by the frequency difference between the two peaks forming the primary peak pair and the frequency at the center of the two peaks, respectively.

In addition, the method of conducting a RD 3DHA,CA,(CO),N,HN NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cαi−1and15Niin a phase sensitive manner in t1(13Cα) and t2(15N) and the chemical shift value of1HNiin t3(1HN), and (2) to avoid cosine modulating the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of1Hαi−1for the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with an additional peak located centrally between two peaks forming the primary peak pair which measures the chemical shift value of13Cαi−1along ω1(13Cα). That additional peak can be derived from13Cαnuclear spin polarization. One specific embodiment (3DHACA(CO)NHN) of this method is illustrated inFIG. 1B, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hαi−1magnetization, which is transferred to13Cαi−1, to15Ni, and to1HNi, to generate the NMR signal. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Bto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2B, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the primary peak pair and a second NMR subspectrum derived from the adding which contains the additional peak located centrally between the two peaks forming the primary peak pair.

In addition, the method of conducting a RD 3DHA,CA,(CO),N,HN NMR experiment can involve applying radiofrequency pulses under conditions effective to additionally cosine modulate the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1. Then, the NMR signals are processed to generate a 3D NMR spectrum with two secondary peak pairs where (1) each of the secondary peak pairs is derived from a different one of the peaks of the primary peak pair, and (2) the chemical shift value of13C′i−1is measured along ω1(13Cα) by the frequency difference between the two peaks forming one of the secondary peak pairs. This method can further involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cαi−1and15Niin a phase sensitive manner in t1(13Cα) and t2(15N) and the chemical shift value of1HNiin t3(1HN), (2) to cosine modulate the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of13C′i−1, and (3) to avoid cosine modulating the13Cαi−1chemical shift evolution in t1(13Cα) with the chemical shift evolution of1Hαi−1. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with an additional secondary peak pair located between the two secondary peak pairs which measures the chemical shift values of13C′i−1and13Cαi−1along ω1(13Cα), by the frequency difference between the two peaks forming the additional secondary peak pair and the frequency at the center of the two peaks, respectively. That additional secondary peak pair can be derived from13Cαnuclear spin polarization. One specific embodiment (3DHACA(CO)NHN) of this method is illustrated inFIG. 1B, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hαi−1magnetization, which is transferred to13Cαi−1, to15Ni, and to1HNi, to generate the NMR signal. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Bto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2B, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the two secondary peak pairs and a second NMR subspectrum derived from the adding which contains the additional peak located centrally between the primary peak pair.

In an alternate embodiment, the RD 3DHA,CA,(CO),N,HN NMR experiment can be modified to a RD 2DHA,CA,(CO,N),HN NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of15Nidoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with a peak pair where (1) the chemical shift value of1HNiis measured in a frequency domain dimension, ω2(1HN), and (2) the chemical shift values of1Hαi−1and13Cαi−1are measured in a frequency domain dimension, ω1(13Cα), by the frequency difference between the two peaks forming the primary peak pair and the frequency at the center of the two peaks, respectively.

In an alternate embodiment, the RD 3DHA,CA,(CO),N,UN NMR experiment can be modified to a RD 4DHA,CA,CO,N,HN NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1, occurs under conditions effective to generate NMR signals encoding the chemical shift value of13C′i−1in a phase sensitive manner in an indirect time domain dimension, t4(13C′). Then, the NMR signals are processed to generate a four dimensional (4D) NMR spectrum with a peak pair where (1) the chemical shift values of15Ni,1HNiand13C′i−1are measured in three frequency domain dimensions, ω2(15N), ω3(1HN), and ω4(13C′), respectively, and (2) the chemical shift values of1Hαi−1and13Cαi−1are measured in a frequency domain dimension, ω1(13Cα), by the frequency difference between the two peaks forming the peak pair and the frequency at the center of the two peaks, respectively.

The 3DH,C,(C-TOCSY-CO),N,HN Experiment

The present invention also relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)H,C,(C-TOCSY—CO),N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having two consecutive amino acid residues, i−1 and i: (1) aliphatic protons of amino acid residue i−1,1Halii−1; (2) aliphatic carbons of amino acid residue i−1,13Calii−1; (3) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (4) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Halii−1and13Calii−1of amino acid residue i−1 are connected to the chemical shift evolutions of15Niand1HNiof amino acid residue i, under conditions effective (1) to generate a NMR signal encoding the chemical shifts of13Calii−1and15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cali) and t2(15N), respectively, and the chemical shift of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the chemical shift evolutions of13Calii−1in t1(13Cali) with the chemical shift evolutions of1Halii−1. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) the chemical shift values of1Halii−1and13Calii−1are measured in a frequency domain dimension, ω1(13Cali), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

In addition, the method of conducting a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Calii−1and15Niin a phase sensitive manner in t1(13Cali) and t2(15N) and the chemical shift value of1HNiin t3(1HN), and (2) to avoid cosine modulating the chemical shift evolutions of13Calii−1in t1(13Cali) with the chemical shift evolution of1Hαi−1for the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with additional peaks located centrally between two peaks forming the peak pairs which measure the chemical shift values of13Calii−1along ω1(13Cali). Those additional peaks can be derived from13Calinuclear spin polarization. One specific embodiment (3DHC-(C-TOCSY—CO)NHN) of this method is illustrated inFIG. 1C, where the applying radiofrequency pulses effects a nuclear spin polarization transfer, where a radiofrequency pulse is used to create transverse1Halii−1magnetization, and1Halii−imagnetization is transferred to13Calii−1, to13Cαi−1, to13C′i−1, to15Ni, and to1HNi, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Cto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2C, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pairs, and a second NMR subspectrum derived from the adding which contains the additional peaks located centrally between the two peaks forming the peak pairs.

In an alternate embodiment, the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment can be modified to a RD 2DH,C,(C-TOCSY—CO,N),HN NMR experiment, which involves applying radiofrequency pulses so that the chemical shift evolution of15Nidoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with peak pairs where (1) the chemical shift value of1HNiis measured in a frequency domain dimension, ω2(1HN), and (2) the chemical shift values of1Halii−1and13Calii−1are measured in a frequency domain dimension, ω1(13Cali), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

In an alternate embodiment, the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment can be modified to a RD 4DH,C,(C-TOCSY),CO,N,HN NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1, occurs under conditions effective to generate NMR signals encoding the chemical shift value of13C′i−1in a phase sensitive manner in an indirect time domain dimension, t4(13C′). Then, the NMR signals are processed to generate a four dimensional (4D) NMR spectrum with variant peak pairs where (1) the chemical shift values of15Ni,1HNiand13C′i−1are measured in three frequency domain dimensions, ω2(15N), ω3(1HN), and ω4(13C′), respectively, and (2) the chemical shift values of1Halii−1and13Calii−1are measured in a frequency domain dimension, ω1(13Cali), by the frequency differences between the two peaks forming the variant peak pairs and the frequencies at the center of the two peaks, respectively.

The 3DHα/β,Cα/β,CO,HA Experiment

Another aspect of the present invention relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)Hα/β,Cα/β,CO,HA nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,1Hβi; (2) a β-carbon of amino acid residue i,13Cβi; (3) an α-proton of amino acid residue i,1Hαi; (4) an α-carbon of amino acid residue i,13Cαi; and (5) a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαi,1Hβi,13Cαi, and13Cβiare connected to the chemical shift evolution of13C′i, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi,13Cβiand13C′iin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα/β) and t2(13C′), respectively, and the chemical shift value of1Hαiin a direct time domain dimension, t3(1Hα), and (2) to cosine modulate the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβi, respectively. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13C′iand1Hαiare measured in two frequency domain dimensions, ω2(13C′) and ω3(1Hα), respectively, and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between the two peaks forming the peak pairs, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

In addition, the method of conducting a RD 3DHα/β,Cα/β,CO,HA NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cαi,13Cβiand15Niin a phase sensitive manner in t1(13Cα/β) and t2(15N) and the chemical shift value of1Hαiin t3(1Hα), and (2) to avoid cosine modulating the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβifor the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with additional peaks located centrally between two peaks forming the peak pairs which measure the chemical shift values of13Cαiand13Cβialong ω1(13Cα/β). Those additional peaks can be derived from13Cαand13Cβnuclear spin polarization. One specific embodiment (3DHα/βCα/βCOHA) of this method is illustrated inFIG. 1E, where the applying radiofrequency pulses effects a nuclear spin polarization transfer, where a radiofrequency pulse is used to create transverse1Hαiand1Hβimagnetization, and1Hαiand1Hβipolarization is transferred to13Cαiand13Cβi, to13C′i, and back to1Hαi, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Eto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2E, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pairs, and a second NMR subspectrum derived from the adding which contains the additional peaks located centrally between the two peaks forming the peak pairs.

In an alternate embodiment, the RD 3DHα/β,Cα/β,CO,HA NMR experiment can be modified to a RD 2DHα/β,Cα/β,(CO),HA NMR experiment, which involves applying radiofrequency pulses so that the chemical shift evolution of13C′idoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with peak pairs where (1) the chemical shift value of1Hαiis measured in a frequency domain dimension, ω2(1Hα), and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between two peaks forming the peak pairs, respectively, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

The 3DHα/β,Cα/β,N,HN Experiment

A further aspect of the present invention relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)Hα/β,Cα/β,N,HN nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule having an amino acid residue, i: (1) a β-proton of amino acid residue i,1Hβi; (2) a β-carbon of amino acid residue i,13Cβi; (3) an α-proton of amino acid residue i,1Hβi; (4) an α-carbon of amino acid residue i,13Cαi; (5) a polypeptide backbone amide nitrogen of amino acid residue i,15Ni; and (6) a polypeptide backbone amide proton of amino acid residue i,1HNi. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hαi,1Hβi,13Cαi, and13Cβiare connected to the chemical shift evolutions of15Niand1HNi, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cαi,13Cβiand15Niin a phase sensitive manner in two indirect time domain dimensions, t1(13Cα/β) and t2(15N), respectively, and the chemical shift value of1HNiin a direct time domain dimension, t3(1HN), and (2) to cosine modulate the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβi, respectively. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of15Niand1HNiare measured in two frequency domain dimensions, ω2(15N) and ω3(1HN), respectively, and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between the two peaks forming the peak pairs, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

In addition, the method of conducting a RD 3DHα/β,Cα/β,N,HN NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cαi,13Cβiand15Niin a phase sensitive manner in t1(13Cα/β) and t2(15N) and the chemical shift value of1HNiin t3(1HN), and (2) to avoid cosine modulating the chemical shift evolutions of13Cαiand13Cβiin t1(13Cα/β) with the chemical shift evolutions of1Hαiand1Hβifor the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with additional peaks located centrally between two peaks forming the peak pairs which measure the chemical shift values of13Cαiand13Cβialong ω1(13Cα/β). Those additional peaks can be derived from13Cαand13Cβnuclear spin polarization. One specific embodiment (3DHα/βCα/βNHN) of this method is illustrated inFIG. 1F, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hαiand1Hβimagnetization, and1Hαiand1Hβimagnetization is transferred to13Cαiand13Cβi, to15Ni, and to1HNi, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Fto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2F, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pairs, and a second NMR subspectrum derived from the adding which contains the additional peaks located centrally between the two peaks forming the peak pairs.

In an alternate embodiment, the RD 3DHα/β,Cα/β,N,HN NMR experiment can be modified to a RD 2DHα/β,Cα/β,(N),HN NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of15Nidoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with peak pairs where (1) the chemical shift value of1HNiis measured in a frequency domain dimension, ω2(1HN), and (2) (i) the chemical shift values of1Hαiand1Hβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequency differences between the two peaks forming the peak pairs, and (ii) the chemical shift values of13Cαi, and13Cβiare measured in a frequency domain dimension, ω1(13Cα/β), by the frequencies at the center of the two peaks forming the peak pairs.

The 3DH,C,C,H—COSY Experiment

The present invention also relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)H,C,C,H—COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm,1Hn, and13Cnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups that are linked by a single covalent carbon—carbon bond in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolutions of1Hnand13Cn, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in two indirect time domain dimensions, t1(13Cm) and t2(13Cn), respectively, and the chemical shift value of1Hnin a direct time domain dimension, t3(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13Cnand1Hnare measured in two frequency domain dimensions, ω2(13Cn) and ω3(1Hn), respectively, and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

In addition, the method of conducting a RD 3DH,C,C,H—COSY NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in t1(13Cm) and t2(13Cn) and the chemical shift value of1Hnin t3(1H), and (2) to avoid cosine modulating the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hmfor the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with additional peaks located centrally between two peaks forming the peak pairs which measure the chemical shift value of13Cmalong ω1(13Cm). Those additional peaks can be derived from13Cmnuclear spin polarization. One specific embodiment (3DHCCH—COSY) of this method is illustrated inFIG. 1H, where the applying radiofrequency pulses effects a nuclear spin polarization transfer according toFIG. 1H, where a radiofrequency pulse is used to create transverse1Hmmagnetization, and1Hmmagnetization is transferred to13Cm, to13Cn, and to1Hn, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Hto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2H, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pairs, and a second NMR subspectrum derived from the adding which contains the additional peaks located centrally between the two peaks forming the peak pairs.

In an alternate embodiment, the RD 3DH,C,C,H—COSY NMR experiment can be modified to a RD 2DH,C,(C),H—COSY NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of13Cndoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with peak pairs where (1) the chemical shift value of1Hnis measured in a frequency domain dimension, ω2(1Hn), and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

The 3DH,C,C,H-TOCSY Experiment

Another aspect of the present invention relates to a method of conducting a reduced dimensionality (RD) three-dimensional (3D)H,C,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm,1Hn, and13Cnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups that may or may not be linked by a single covalent carbon-carbon bond in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolutions of1Hnand13Cn, under conditions effective (1) to generate NMR signals encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in two indirect time domain dimensions, t1(13Cm) and t2(13Cn), and the chemical shift value of1Hnin a direct time domain dimension, t3(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 3D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift values of13Cnand1Hnare measured in two frequency domain dimensions, ω2(13Cn) and ω3(1Hn), respectively, and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

In addition, the method of conducting a RD 3DH,C,C,H-TOCSY NMR can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift values of13Cmand13Cnin a phase sensitive manner in t1(13Cm) and t2(13Cn) and the chemical shift value of1Hnin t3(1Hn), and (2) to avoid cosine modulating the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hmfor the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 3D NMR spectrum with additional peaks located centrally between two peaks forming the peak pairs which measure the chemical shift value of13Cmalong ω1(13Cm). Those additional peaks can be derived from13Cmnuclear spin polarization. One specific embodiment (3DHCCH-TOCSY) of this method is illustrated inFIG. 1I, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hmmagnetization, and1Hmmagnetization is transferred to13Cm, to13Cn, and to1Hn, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Ito generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2I, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pairs, and a second NMR subspectrum derived from the adding which contains the additional peaks located centrally between the two peaks forming the peak pairs.

In an alternate embodiment, the RD 3DH,C,C,H-TOCSY NMR experiment can be modified to a RD 2DH,C,(C),H-TOCSY NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of13Cndoes not occur. Then, the NMR signals are processed to generate a two dimensional (2D) NMR spectrum with peak pairs where (1) the chemical shift value of1Hnis measured in a frequency domain dimension, ω2(1Hn), and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

The 2DHB,CB,(CG,CD),HD Experiment

A further aspect of the present invention relates to a method of conducting a reduced dimensionality (RD) two-dimensional (2D)HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for the following nuclei of a protein molecule: (1) a β-proton of an amino acid residue with an aromatic side chain,1Hβ; (2) β-carbon of an amino acid residue with an aromatic side chain,13Cβ; and (3) a δ-proton of an amino acid residue with an aromatic side chain,1Hδ. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hβand13Cβare connected to the chemical shift evolution of1Hδ, under conditions effective (1) to generate NMR signals encoding the chemical shift value of13Cβin a phase sensitive manner in an indirect time domain dimension, t1(13Cβ), and the chemical shift value of1Hδin a direct time domain dimension, t2(1Hδ), and (2) to cosine modulate the chemical shift evolution of13Cβin t1(13Cβ) with the chemical shift evolution of1Hβ. Then, the NMR signals are processed to generate a 2D NMR spectrum with a peak pair derived from the cosine modulating where (1) the chemical shift value of1Hδis measured in a frequency domain dimension, ω2(1Hδ), and (2) the chemical shift values of1Hβand13Cβare measured in a frequency domain dimension, ω1(13Cβ), by the frequency difference between the two peaks forming the peak pair and the frequency at the center of the two peaks, respectively.

In addition, the method of conducting a RD 2DHB,CB,(CG,CD),HD NMR experiment can involve applying radiofrequency pulses under conditions effective (1) to generate an additional NMR signal encoding the chemical shift value of13Cβin a phase sensitive manner in t1(13Cβ) and the chemical shift value of1Hδin t2(1Hδ), and (2) to avoid cosine modulating the chemical shift evolution of13Cβin t1(13Cβ) with the chemical shift evolution of1Hβfor the additional NMR signal. Then, the NMR signals and the additional NMR signal are processed to generate a 2D NMR spectrum with an additional peak located centrally between the two peaks forming the peak pair which measure the chemical shift value of13Cβalong ω1(13C). That additional peak can be derived from13Cβnuclear spin polarization. One specific embodiment (2DHBCB(CGCD)HD) of this method is illustrated inFIG. 1J, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hβmagnetization, and1Hβmagnetization is transferred to13Cβ, to13Cδ, and to1Hδ, where the NMR signal is detected. Another specific embodiment of this method involves applying radiofrequency pulses by (1) applying a first set of radiofrequency pulses according to the scheme shown inFIG. 2Jto generate a first NMR signal, and (2) applying a second set of radiofrequency pulses according to the scheme shown inFIG. 2J, where phase φ1of the first1H pulse is altered by 180° to generate a second NMR signal. Then, prior to the processing, the first NMR signal and the second NMR signal are added and subtracted, whereby the NMR signals are processed to generate a first NMR subspectrum derived from the subtracting which contains the peak pair, and a second NMR subspectrum derived from the adding which contains the additional peak located centrally between the two peaks forming the peak pair.

In an alternate embodiment, the RD 2DHB,CB,(CG,CD),HD NMR experiment can be modified to a RD 3DHB,CB,(CG),CD,HD NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of a δ-carbon of an amino acid residue with an aromatic side chain,13Cδoccurs under conditions effective to generate NMR signals encoding the chemical shift value of13Cδin a phase sensitive manner in an indirect time domain dimension, t3(13Cδ). Then, the NMR signals are processed to generate a three dimensional (3D) NMR spectrum with a peak pair where (1) the chemical shift values of1Hδand13Cδare measured in two frequency domain dimensions, ω2(1Hδ) and ω3(13Cδ), respectively, and (2) the chemical shift values of1Hβand13Cβare measured in a frequency domain dimension, ω1(13Cβ), by the frequency difference between the two peaks forming the peak pair and the frequency at the center of the two peaks, respectively.

In an alternate embodiment, the RD 2DHB,CB,(CG,CD),HD NMR experiment can be modified to a RD 3DHB,CB,CG,(CD),HD NMR experiment which involves applying radiofrequency pulses so that the chemical shift evolution of a γ-carbon of an amino acid residue with an aromatic side chain,13Cγoccurs under conditions effective to generate NMR signals encoding the chemical shift value of13Cγin a phase sensitive manner in an indirect time domain dimension, t3(13Cγ), and said processing the NMR signals generates a three dimensional (3D) NMR spectrum with a peak pair wherein (1) the chemical shift values of1Hδand13Cγare measured in two frequency domain dimensions, ω2(1Hδ) and ω3(13Cγ), respectively, and (2) the chemical shift values of1Hβand13Cβare measured in a frequency domain dimension, ω1(13Cβ), by the frequency difference between the two peaks forming said peak pair and the frequency at the center of the two peaks, respectively.

The 2DH,C,H—COSY Experiment

The present invention also relates to a method of conducting a reduced dimensionality (RD) two-dimensional (2D)H,C,H—COSY nuclear magnetic resonance (NMR) experiment by measuring the chemical shift values for1Hm,13Cm, and1Hnof a protein molecule where m and n indicate atom numbers of two CH, CH2or CH3groups in an amino acid residue. The method involves providing a protein sample and applying radiofrequency pulses to the protein sample which effect a nuclear spin polarization transfer where the chemical shift evolutions of1Hmand13Cmare connected to the chemical shift evolution of1Hn, under conditions effective (1) to generate NMR signals encoding the chemical shift value of13Cmin a phase sensitive manner in an indirect time domain dimension, t1(13Cm), and the chemical shift value of1Hnin a direct time domain dimension, t2(1Hn), and (2) to cosine modulate the chemical shift evolution of13Cmin t1(13Cm) with the chemical shift evolution of1Hm. Then, the NMR signals are processed to generate a 2D NMR spectrum with peak pairs derived from the cosine modulating where (1) the chemical shift value of1Hnis measured in a frequency domain dimension, ω2(1Hn), and (2) the chemical shift values of1Hmand13Cmare measured in a frequency domain dimension, ω1(13Cm), by the frequency differences between the two peaks forming the peak pairs and the frequencies at the center of the two peaks, respectively.

One specific embodiment (2D1H-TOCSY—HCH—COSY) of this method is illustrated inFIG. 1K, where the applying radiofrequency pulses effects a nuclear spin polarization transfer where a radiofrequency pulse is used to create transverse1Hmmagnetization, and1Hmpolarization is transferred to13Cm, to1Hm, and to1Hn, where the NMR signal is detected. Although the specific embodiment illustrated inFIG. 1Kshows this method applied to an amino acid residue with an aromatic side chain, this method also applies to amino acid residues with aliphatic side chains. Another specific embodiment of this method involves applying radiofrequency pulses according to the scheme shown inFIG. 2K.

FIG. 3outlines which chemical shifts are correlated in the various NMR experiments described above.

Combinations of RD NMR Experiments

Accordingly, a suite of multidimensional RD NMR experiments enables one to devise strategies for RD NMR-based HTP resonance assignment of proteins.

Thus, another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an α-proton,1Hα, an α-carbon,13Cα, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN,of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DHA,CA,(CO),N,HN NMR experiment to measure and connect chemical shift values of the α-proton of amino acid residue i−1,1Hαi−1, the α-carbon of amino acid residue i−1,13Cαi−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3D HNNCAHANMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hα,13Cα,15N, and1HNare obtained by (i) matching the chemical shift values of1Hαi−1and13Cαi−1with the chemical shift values of1Hαiand13Cαi, (ii) using the chemical shift values of1Hαi−1and13Cαi−1to identify the type of amino acid residue i−1 (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et al.,J. Biomol. NMR,3: 185–204 (1993), which are hereby incorporated by reference in their entirety), and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements (such as α-helices and β-sheets) within the polypeptide chain (Spera et al.,J. Am. Chem. Soc.,113:5490–5492 (1991); Wishart et al., Biochemistry, 31:1647–1651, which are hereby incorporated by reference in their entirety).

In one embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift value of13C′i−1, are obtained by matching the chemical shift value of13Cαimeasured by the RD 3D HNN<CO,CA> NMR experiment with the sequentially assigned chemical shift values of13Cα,15N, and1HNmeasured by the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to (i) a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,1Hβi, the β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i, and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of13C′i, the α-carbon of amino acid residue i+1,13Cαi+1, the polypeptide backbone amide nitrogen of amino acid residue i+1,15Ni+1, and the polypeptide backbone amide proton of amino acid residue i+1,1HNi+1. Then, sequential assignments are obtained by matching the chemical shift value of13C′, measured by the RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of13C′imeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to measure and connect the chemical shift values of aliphatic protons (including α-, β-, and γ-protons) of amino acid residue i−1,1Halii−1, aliphatic carbons (including α-, β-, and γ-carbons) of amino acid residue i−1,13Calii−1,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Halii−1and13Calii−1for amino acid residues i having unique pairs of15Niand1HNichemical shift values are obtained by matching the chemical shift values of1Hαand13Cαmeasured by said RD 3D HNNCAHANMR experiment and RD 3DHA,CA,(CO),N,HN NMR experiment with the chemical shift values of1Hαi−1and13Cαi−1measured by said RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and using the1Halii−1and13Calii−1chemical shift values to identify the type of amino acid residue i−1.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3DH,C,C,H—COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of1Haliiand13Caliiof amino acid residue i. Then, sequential assignments of the chemical shift values of1Haliiand13Calii, the chemical shift values of a γ-proton,1Hγi, and a γ-carbon,13Cγi, in particular, are obtained by (i) matching the chemical shift values of1Hαiand13Cαimeasured using the RD 3DH,C,C,H—COSY NMR experiment or the RD 3DH,C,C,H-TOCSY RD NMR experiment with the chemical shift values of1Hαiand13Cαmeasured by the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment and (ii) using the chemical shift values of1Haliand13Cali, the chemical shift values of1Hγiand13Cγiin particular, to identify the type of amino acid residue i.

In yet another embodiment, this method involves, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, further subjecting the protein sample to a RD 3DHα/βCα/β(CO)NHN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i−1,1Hβi−1, the β-carbon of amino acid residue i−1,13Cβi−1,1Hαi−1,13Cαi−1,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hβand13Cβare obtained by using the chemical shift values of1Hβi−1and13Cβi−1to identify the type of amino acid residue i−1.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment, be further subjected to a RD 3D Hα/β, Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,1Hβi, the β-carbon of amino acid residue i,13Cβi,1Hαi,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. Then, sequential assignments of the chemical shift value of13C′iare obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment with the sequentially assigned chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNmeasured by the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαiwith the chemical shift values of1Hβi−1,13Cβi−1,1Hαi−1, and13Cαi−1measured by the RD 3DHα/βCα/β(CO)NHN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of13Cβi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of13Cβiand13Cαimeasured by said 3D HNNCACB NMR experiment with the chemical shift values of13Cβi−1and13Cαi−1measured by the RD 3DHα/βCα/β(CO)NHN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of1Hβi−1,13Cβi−1, and a δ-proton of amino acid residue I−1 with an aromatic side chain,1Hδi−1. Then, sequential assignments are obtained by matching (i) the chemical shift values of1Hβi−1and13Cβi−1measured by said RD 2DHB,CB,(CG,CD),HD NMR experiment with the chemical shift values of1Hβand13Cβmeasured by the RD 3DHα/βCα/β(CO)NHN NMR experiment, (ii) using the chemical shift values to identify amino acid residue i as having an aromatic side chain, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along the polypeptide chain.

In another embodiment, the protein sample could, in addition to the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment, be further subjected to a RD 3DH,C,C,H—COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons (including α-, β-, and γ-protons) of amino acid residue i,1Halii, and aliphatic carbons (including α-, β-, and γ-carbons) of amino acid residue i,13Calii, of amino acid residue i. Then, sequential assignments of the chemical shift values of1Haliiand13Calii, the chemical shift values of a γ-proton,1Hγ, and a γ-carbon,13Cγ, in particular, are obtained by (i) matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured using the RD 3DH,C,C,H—COSY NMR experiment or the RD 3DH,C,C,H-TOCSY RD NMR experiment with the chemical shift values of1Hβi,13Cβi,1Hαi, and13Caimeasured by the RD 3DHA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHANMR experiment, and the RD 3DHα/βCα/β(CO)NHN NMR experiment and (ii) using the chemical shift values of1Haliand13Cali, the chemical shift values of1Hγand13Cγin particular, to identify the type of amino acid residue i.

Yet another aspect of the present invention relates to a method for sequentially assigning chemical shift values of a β-proton,1Hβ, a β-carbon,13Cβ, an α-proton,1Hα, an α-carbon,13Cα, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HNi, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DHα/βCα/β(CO)NHN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i−1,1Hβi−1, the β-carbon of amino acid residue i−1,13Cβi−1, the α-proton of amino acid residue i−1,1Hαi−1, the α-carbon of amino acid residue i−1,13Cαi−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure and connect the chemical shift values of the β-proton of amino acid residue i,1Hβi, the β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNare obtained by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,1Hα/βi−1, and the chemical shift values of the α- and β-carbons of amino acid residue i−1,13Cα/βi−1, with1Hα/βiand13Cα/βi, (ii) using1Hα/βi−1and13Cα/βi−1to identify the type of amino acid residue i−1 (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et al.,J. Biomol. NMR,3: 185–204 (1993), which are hereby incorporated by reference in their entirety), (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain (Spera et al.,J. Am. Chem. Soc.,113:5490–5492 (1991); Wishart et al., Biochemistry, 31:1647–1651, which are hereby incorporated by reference in their entirety).

In one embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cαβ, N,HN NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment (i) to measure and connect chemical shift values of1Hαi−1,13Cαi−1,15Ni, and1HNiand (ii) to distinguish between NMR signals for1Hα/13Cαand1Hβ/13Cβmeasured in the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/βN,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαl,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. Then, sequential assignments of the chemical shift value of13C′iare obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment with the sequentially assigned chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNmeasured by the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift value of13C′i−1are obtained by matching the chemical shift value of13Cαimeasured by the RD 3D HNN<CO,CA> NMR experiment with the sequentially assigned chemical shift values of13Cα,15N, and1HNmeasured by the RD 3DHα/βCα/β(CO)NHN NMR experiment and RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cαβ,N,HN NMR experiment, be further subjected to (i) a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαi,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′iand (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of13C′i, the α-carbon of amino acid residue i+1,13Cαi+1, the polypeptide backbone amide nitrogen of amino acid residue i+1,15Ni+1, and the polypeptide backbone amide proton of amino acid residue i+1,1HNi+1. Then, sequential assignments are obtained by matching the chemical shift value of13C′imeasured by said RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of13C′imeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to measure and connect the chemical shift values of1Halii−1,13Calii−1,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Halii−1and13Calii−1for amino acid residues i having unique pairs of15Niand1HNichemical shift values are obtained by matching the chemical shift values of1Hβ,13Cβ,1Hα, and13Cαmeasured by the RD 3DHα/βCα/β(C))NHN NMR experiment and RD 3DHα/β,Cα/β,N,HN NMR experiment with the chemical shift values of1Hβi−1,13Cβi−1,1Hαi−1, and13Cαi−1measured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and using the1Halii−1, and13Calii−1chemical shift values to identify the type of amino acid residue i−1.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cαβ,N,HN NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of13Cβi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of13Cβiand13Cαimeasured by said 3D HNNCACB NMR experiment with the chemical shift values of13Cβi−1and13Cαi−1measured by the RD 3DHα/βCα/β(CO)NHN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cαβ,N,HN NMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi, and a δ-proton of amino acid residue i with an aromatic side chain,1Hδi. Then, sequential assignments are obtained by (i) matching the chemical shift values of1Hβiand13Cβimeasured by said RD 2DHB,CB,(CG,CD),HD NMR experiment with the chemical shift values of1Hβand13Cβmeasured by the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, (ii) using the chemical shift values to identify amino acid residue i as having an aromatic side chain, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along the polypeptide chain (Spera et al.,J. Am. Chem. Soc.,113:5490–5492 (1991); Wishart et al., Biochemistry, 31:1647–1651, which are hereby incorporated by reference in their entirety).

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DH,C,C,H—COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,1Halii, and aliphatic carbons of amino acid residue i,13Calii, of amino acid residue i. Then, sequential assignments of the chemical shift values of1Haliiand13Calii, the chemical shift values of a γ-proton,1Hγi, and a γ-carbon,13Cγi, in particular, are obtained by (i) matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured using the RD 3DH,C,C,H—COSY NMR experiment or the RD 3DH,C,C,H-TOCSY RD NMR experiment with the chemical shift values of1Hβ,13Cβ,1Hα, and13Cαmeasured by the RD 3DHα/βCα/β(CO)NHN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, and (ii) using the chemical shift values of1Haliiand13Calii, the chemical shift values of1Hγiand13Cγiin particular, to identify the type of amino acid residue i.

A further aspect of the present invention involves a method for sequentially assigning the chemical shift values of aliphatic protons,1Hali, aliphatic carbons,13Cali, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN,of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,1Halii−1, the aliphatic carbons of amino acid residue i−1,13Calii−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure the chemical shift values of the β-proton of amino acid residue i,1Hβi, the β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hali,13Cali,15N, and1HNare obtained by (i) matching the chemical shift values of the α- and β-protons of amino acid residue i−1,1Hα/βi−1and the α- and β-carbons of amino acid residue i−1,13Cα/βi−1with the chemical shift values of1Hα/β1and13Cα/βiof amino acid residue i, (ii) using the chemical shift values of1Halii−1and13Calii−1to identify the type of amino acid residue i−1 (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et al.,J. Biomol. NMR,3: 185–204 (1993), which are hereby incorporated by reference in their entirety), and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain (Spera et al.,J. Am. Chem. Soc.,113:5490–5492 (1991); Wishart et al., Biochemistry, 31:1647–1651, which are hereby incorporated by reference in their entirety).

In one embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαi,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. Then, sequential assignments of the chemical shift value of13C′iare obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment with the sequentially assigned chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNmeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C, (C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,N,HN,NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′−1,13Cαi,15N1, andiHNi. Then, sequentail assignments of the chemical shift value of13C′i−1are obtained by matching the chemical shift value of13Cαimeasured by the RD 3D HNN<CO,CA> NMR experiment with the sequentially assigned chemical shift values of13Cα,15N, and1HNmeasured by the RD 3DH,C, (C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experimnet, be further subjected to (i) a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαi,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i, and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of13C′i, the α-carbon of amino acid residue i+1,13Cαi+1, the polypeptide backbone amide nitrogen of amino acid residue i+1,15Ni+1, and the polypeptide backbone amide proton of amino acid residue i+1,1HNi+1. Then, sequential assignments are obtained by matching the chemical shift value of13C′imeasured by the RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of13C′imeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DHα/βCα/β(CO)NHN NMR experiment (i) to measure and connect the chemical shift values of1Hα/βi−1,13Cα/βi−1,15Ni, and1HNi, and (ii) to identify NMR signals for1Hα/βi−1,13Cα/βi−1,15Ni, and1HNiin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment (i) to measure and connect chemical shift values of1Hαi−1,13Cαi−1,15Ni, and1HNiand (ii) to identify NMR signals for1Hαand13Cαin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to measure and connect the chemical shift value of13Cβi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of13Cβiand13Cαimeasured by said 3D HNNCACB NMR experiment with the chemical shift values of13Cβi−1and13Cαi−1measured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi, and a δ-proton of amino acid residue i with an aromatic side chain,1Hδi. Then, sequential assignments are obtained by matching the chemical shift values of1Hβiand13Cβimeasured by said RD 2DHB,CB,(CG,CD)ND NMR experiment with the chemical shift values of1Hβand13Cβmeasured by the RD 3DHα/β,Cα/β,N,HN NMR experiment and the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, using the chemical shift values to identify amino acid residue i as having an aromatic side chain, and mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and locating amino acid residues with aromatic side chains along the polypeptide chain.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3DHα/β,Cα/β,N,HN NMR experiment, be further subjected to a RD 3DH,C, C,H—COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,1Halii, and aliphatic carbons of amino acid residue i,13Calii. Then, sequential assignments of the chemical shift values of1Haliiand13Calii, the chemical shift values of a γ-proton,1Hγi, and a γ-carbon,13Cγi, in particular, are obtained by (i) matching the chemical shift values of1Haliiand13Caliimeasured using the RD 3DH,C,C,H—COSY NMR experiment or the RD 3DH,C,C,H-TOCSY NMR experiment with the chemical shift values of1Haliand13Calimeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and RD 3DHα/β,Cα/β,N,HN NMR experiment, and (ii) using the chemical shift values of1Haliiand13Calii, the chemical shift values of1Hγiand13Cγiin particular, to identify the type of amino acid residue i.

The present invention also relates to a method for sequentially assigning chemical shift values of aliphatic protons,1Hali, aliphatic carbons,13Cali, a polypeptide backbone amide nitrogen,15N, and a polypeptide backbone amide proton,1HN, of a protein molecule. The method involves providing a protein sample and conducting a set of reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to measure and connect the chemical shift values of the aliphatic protons of amino acid residue i−1,1Halii−1, the aliphatic carbons of amino acid residue i−1,13Calii−1, the polypeptide backbone amide nitrogen of amino acid residue i,15Ni, and the polypeptide backbone amide proton of amino acid residue i,1HNiand (2) a RD 3D HNNCAHANMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift values of1Hali,13Cali,15N, and1HNare obtained by (i) matching the chemical shift values of the α-proton of amino acid residue i−1,1Hαi−1and the α-carbon of amino acid residue i−1,13Cαi−1with the chemical shift values of1Hαiand13Cαi, (ii) using the chemical shift values of1Halii−1and13Calii−1to identify the type of amino acid residue i−1 (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et al.,J. Biomol. NMR,3: 185–204 (1993), which are hereby incorporated by reference in their entirety), and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using the chemical shift values to locate secondary structure elements within the polypeptide chain (Spera et al.,J. Am. Chem. Soc.,113:5490–5492 (1991); Wishart et al., Biochemistry, 31:1647–1651, which are hereby incorporated by reference in their entirety).

In one embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of a β-proton of amino acid residue i,1Hβi, a β-carbon of amino acid residue i,13Cβi,1Hαi,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i. Then, sequential assignments of the chemical shift value of13C′iare obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment with the sequentially assigned chemical shift values of1Hβ,13Cβ,1Hα,13Cα,15N, and1HNmeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of a polypeptide backbone carbonyl carbon of amino acid residue i−1,13C′i−1,13Cαi,15Ni, and1HNi. Then, sequential assignments of the chemical shift value of13C′i−1are obtained by matching the chemical shift value of13Cαimeasured by the RD 3D HNN<CO,CA> NMR experiment with the sequentially assigned chemical shift values of13Cα,15N, and1HNmeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to (i) a RD 3DHα/β,Cα/β,CO,HA NMR experiment to measure and connect the chemical shift values of a β-proton of amino acid residue i,1Hβi, a β-carbon of amino acid residue i,13Cβi, the α-proton of amino acid residue i,1Hαi, the α-carbon of amino acid residue i,13Cαi, and a polypeptide backbone carbonyl carbon of amino acid residue i,13C′i, and (ii) a RD 3D HNN<CO,CA> NMR experiment to measure and connect the chemical shift values of13C′i, an α-carbon of amino acid residue i+1,13Cαi+1, a polypeptide backbone amide nitrogen of amino acid residue i+1,15Ni+1, and the polypeptide backbone amide proton of amino acid residue i+1,1HNi+1. Then, sequential assignments are obtained by matching the chemical shift value of13C′imeasured by the RD 3D HNN<CO,CA> NMR experiment with the chemical shift value of13C′imeasured by the RD 3DHα/β,Cα/β,CO,HA NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3D Hα/βCα/β(CO)NHN NMR experiment (i) to measure and connect the chemical shift values of the α- and β-protons of amino acid residue i−1,1Hα/βi−1, the α- and β-carbons of amino acid residue i−1,13Cα/βi−1,15Ni, and1HNi, and (ii) to distinguish NMR signals for the chemical shift values of1Hβi−1,13Cβi−1,1Hαi−1, and13Cαi−1measured by the RD 3DHα/βCα/β(CO)NHN NMR experiment from NMR signals for the chemical shift values of1Halii−1and13Calii−1other than1Hα/βi−1and13Cα/βi−1measured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3DHα/β,Cα/β,N,HN NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi,1Hαi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by said RD 3DHα/β,Cα/β,N,HN NMR experiment with the chemical shift values of1Hβi−1,—Cβi−,1Hαi−1, and13Cαi−1measured by the RD 3DH,C(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a 3D HNNCACB NMR experiment to measure and connect the chemical shift values of13Cβi,13Cαi,15Ni, and1HNi. Then, sequential assignments are obtained by matching the chemical shift values of13Cβiand13Cαimeasured by said 3D HNNCACB NMR experiment with the chemical shift values of13Cβi−1and13Cαi−1measured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 2DHB,CB,(CG,CD),HD NMR experiment to measure and connect the chemical shift values of1Hβi,13Cβi, and a δ-proton of amino acid residue i with an aromatic side chain,1Hδi. Then, sequential assignments are obtained by matching the chemical shift values of1Hβiand13Cβimeasured by said RD 2DHB,CB,(CG,CD),HD NMR experiment with the chemical shift values of1Hβand13Cβmeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, using the chemical shift values to identify amino acid residue i as having an aromatic side chain, and mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and by locating amino acid residues with aromatic side chains along the polypeptide chain.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, be further subjected to a RD 3DH,C,C,H—COSY NMR experiment or a RD 3DH,C,C,H-TOCSY NMR experiment to measure and connect the chemical shift values of aliphatic protons of amino acid residue i,1Halii, and aliphatic carbons of amino acid residue i,13Calii. Then, sequential assignments of the chemical shift values of1Haliiand13Calii, the chemical shift values of a γ-proton,1Hγi, and a γ-carbon,13Cγi, in particular, are obtained by (i) matching the chemical shift values of1Haliand13Calimeasured using the RD 3DH,C,C,H—COSY NMR experiment or the RD 3DH,C,C,H-TOCSY NMR experiment with the chemical shift values of1Hβi,13Cβi,1Hαi, and13Cαimeasured by the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the RD 3D HNNCAHANMR experiment, and (ii) using the chemical shift values of1Haliiand13Calii, the chemical shift values of1Hγiand13C65iin particular, to identify the type of amino acid residue i.

Another aspect of the present invention involves a method for obtaining nearly complete assignments of chemical shift values of1H,13C and15N of a protein molecule (excluding only chemical shift values of13Cδand15Nε2of glutamines, of13Cγand15Nδ2of asparagines, of13Cε3,1Hε3,13Cζ2,1Hζ2,13Cζ3,1Hζ3,13Cη2, and1Hη2groups of tryptophans, of13Cεand1Hεof methionines, and of labile sidechain protons that exchange rapidly with the protons of the solvent water) (Yamazaki et al.,J. Am. Chem. Soc.,115:11054–11055 (1993), which is hereby incorporated by reference in its entirety), which are required for the determination of the tertiary structure of a protein in solution (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986), which is hereby incorporated by reference in its entirety). The method involves providing a protein sample and conducting four reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, where (1) a first experiment is selected from the group consisting of a RD three-dimensional (3D)Hα/βCα/β(CO)NHN NMR experiment, a RD 3DHA,CA,(CO),N,HN NMR experiment, and a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment for obtaining sequential correlations of chemical shift values; (2) a second experiment is selected from the group consisting of a RD 3D HNNCAHANMR experiment, a RD 3DHα/β,Cα/β,N,HN NMR experiment, and a RD 3D HNN<CO,CAexperiment for obtaining intraresidue correlations of chemical shift values; (3) a third experiment is a RD 3DH,C,C,H—COSY NMR experiment for obtaining assignments of aliphatic and aromatic sidechain chemical shift values; and (4) a fourth experiment is a RD 2DHB,CB,(CG,CD),HD NMR experiment for obtaining assignments of aromatic sidechain chemical shift values.

In one embodiment of this method, the protein sample could be further subjected to a RD 2DH,C,H—COSY NMR experiment for obtaining assignments of aliphatic and aromatic sidechain chemical shift values.

In another embodiment of this method, the first experiment is the RD 3DHα/βCα/β(CO)NHN NMR experiment and the second experiment is the RD 3D HNNCAHANMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to distinguish between NMR signals for1Hα/13Cαand1Hβ/13Cβfrom the RD 3DHα/βCα/β(CO)NHN NMR experiment.α/β(CO)NHN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment to obtain assignments of chemical shift values of1Haliand13Cali.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,N,HN NMR experiment to obtain assignments of chemical shift values of1Hβand13Cβ.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment and a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of13C′.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aliphatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aromatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DHα/βCα/β(CO)NHN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of13Cβ.

In yet another embodiment of this method, the first experiment is the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the second experiment is the RD 3D HNNCAHANMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for1Hα/13Cαin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,N,HN NMR experiment to obtain assignments of chemical shift values of1Hβand13Cβ.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment and a RD 3DHα/α,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aliphatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aromatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNNCAHANMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of13Cβ.

In yet another embodiment of this method, the first experiment is the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the second experiment is the RD 3DHα/β,Cα/β,N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β, N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for1Hαand13Cαin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β, N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/βCα/β(CO)NHN NMR experiment to identify NMR signals for1Hα/βand13Cα/βin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D HNN<CO,CA> NMR experiment and a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aliphatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aromatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3DHα/β,Cα/β,N,HN NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of13Cβ.

In yet another embodiment of this method, the first experiment is the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment and the second experiment is the RD 3D HNN<CO,CA> NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHA,CA,(CO),N,HN NMR experiment to identify NMR signals for1Hαand13Cαin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/βCα/β(CO)NHN NMR experiment to identify NMR signals for1Hα/βandα/βin the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DHα/β,Cα/β,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons,13C′.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aliphatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3DH,C,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of1H and13C of aromatic sidechains.

In another embodiment, the protein sample could, in addition to the RD 3DH,C,(C-TOCSY—CO),N,HN NMR experiment, RD 3D HNN<CO,CA> NMR experiment, RD 3DH,C,C,H—COSY NMR experiment, and RD 2DHB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of13Cβ.

In addition, the above-described method for obtaining assignments of chemical shift values of1H,13C and15N of a protein molecule can involve further subjecting the protein sample to nuclear Overhauser effect spectroscopy (NOESY) (Wüthrich,NMR of Proteins and Nucleic Acids, Wiley, New York (1986), which is hereby incorporated by reference in its entirety), to NMR experiments that measure scalar coupling constants (Eberstadt et al.,Angew. Chem. Int. Ed. Engl.,34:1671–1695 (1995); Cordier et al.,J. Am. Chem. Soc.,121:1601–1602 (1999), which are hereby incorporated by reference in their entirety), or to NMR experiments that measure residual dipolar coupling constants (Prestegard,Nature Struct. Biol.,5:517–522 (1998); Tjandra et al.,Science,278:1111–1114 (1997), which are hereby incorporated by reference in their entirety), to deduce the tertiary fold or tertiary structure of the protein molecule.

A standard set of nine experiments (labeled with asterisks in Table 2) can be employed for obtaining nearly complete resonance assignments of proteins including aliphatic and aromatic side chain spin systems.

TABLE 2NMR experiments acquiredafor the 8.5 kDa protein “Z-domain”Minimalcmeasurementtime [h]Indirectbtmax; ComplexMeasurementwith/withoutExperimentdimension(s)points [ms]time [h]central peak3D spectra for sequential backbone connectivities:**Hα/βCα/β(CO)NHNω1(13Cα/β)6.3; 959.24.6/2.3ω2(15N)21.5; 28HACA(CO)NHNω1(13Cα)6.5; 545.4/2.7d5.4/2.7ω2(15N)21.5; 28HC(C-TOCSY-CO)NHNdω1(13Cα/β)6.1; 9017.94.5/2.3ω2(15N)21.5; 283D spectra for intraresidual backbone connectivities:**HNNCAHAω1(13Cα)6.6;515.02.5/n.a.ω2(15N)21.5; 28*Hα/βCα/βCOHAω1(13Cα/β)6.3; 9510.05.0/2.5ω2(13C═O)17.8; 32Hα/βCα/βNHNω1(13Cα/β)6.0; 9017.14.3/2.2ω2(15N)21.5; 28*HNNCACBω1(13Cα/β)6.6; 568.0n.a./2.0ω2(15N)21.5; 283D spectrum for intra- and sequential backbone connectivities:*HNN<CO,CA>ω1(13C═O)8.0/16.0e; 545.52.8/n.a.ω2(15N)21.5; 283D spectra for assignment of aliphatic resonances:**HCCH-COSYω1(13C)6.3; 956.23.1/1.6ω2(13C)6.4; 20**HCCH-TOCSYfω1(13C)6.3; 957.03.5/1.7ω2(13C)6.4; 202D spectra for assignment of aromatic resonances:**HBCB(CGCD)HDω1(13C)6.3; 955.30.1/0.05**1H-TOCSY-HCH-COSYfω1(13C)15; 1503.40.2/n.a.a1 mM solution of “Z-domain” ofStaphylococcalprotein A25at T = 25° C. The1H carrier for1H-frequency labeling in the projected “HC”-dimensions was set to 0 ppm relative to DSS. tmaxdenotes the maximal evolution time. Spectra forming a “standard set” that has been inferred from the present study (see text) are labeled with an asterisk (* or **), and those spectra which can be designated a “minimal” set are labeled with a double-asterisk (**).bDirect dimension: tmax= 73 ms/512 complex points.cThe minimal measurement time (rounded) was calculated for the acquisition of a single transient per FID, either with (left number) or without (right number) acquisition of central peaks. Other spectral parameters were assumed to be unchanged. Note that central peak acquisition (Szyperski et al.,J. Am. Chem. Soc., 118:8146–8147 (1996), which is hereby incorporated by reference in its entirety) from13C magnetization requires recording of two data sets that are added and subtracted to generate subspectra I and II.dThe mixing times for the13C-TOCSY relay was set to 14 ms or 21 ms.eThe increment for13Cαchemical shift evolution was scaled (Szyperski et al.,J. Am. Chem. Soc., 115:9307–9308 (1993); Szyperski et al.,J. Magn. Reson., B 108, 197–203 (1995), which are hereby incorporated by reference in their entirety) by a factor of 0.5 relative to the13C═O evolution.fThe mixing time for the1H-TOCSY relay was set to 25 ms.

For larger proteins, complementary recording of highly sensitive 3DHACA(CO)NHN promises (i) to yield spin systems which escape detection inHα/βCα/β(CO)NHN, and (ii) to offer the distinction of α- and β-moiety resonances by comparison withHα/βCα/β(CO)NHN. Furthermore, employment of 50% random fractional protein deuteration (LeMaster,Annu. Rev. Biophys. Biophys. Chem.,19:43–266 (1990); Nietlispach et al.,J. Am. Chem. Soc.,118:407–415 (1996); Shan et al.,J. Am. Chem. Soc.,118:6570–6579 (1996); Leiting et al.,Anal. Biochem.,265:351–355 (1998); Hochuli et al.,J. Biomol. NMR,17:33–42 (2000), which are hereby incorporated by reference in their entirety) in combination with the standard suite of NMR experiments (or transverse relaxation-optimized spectroscopy (TROSY) versions thereof) is attractive. The impact of deuteration for recording 4D Hα/βCα/β(CO)NHN for proteins reorienting with correlation times up to around 20 ns (corresponding to a molecular weight around 30 kDa at ambient T) has been demonstrated (Nietlispach et al.,J. Am. Chem. Soc.,1 18:407–415 (1996), which is hereby incorporated by reference in its entirety). Accordingly, 3DHα/βCα/β(CO)NHN can be expected to maintain its pivotal role for obtaining complete resonance assignments (FIG. 4) for deuterated proteins at least up to about that size. Furthermore, protein deuteration offers the advantage that HNNCACB, which can be expected to become significantly less sensitive than HNNCAHAfor larger non-deuterated systems, (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety) can be kept to recruit13Cβchemical shifts for sequential assignment (Shan et al.,J. Am. Chem. Soc.,118:6570–6579 (1996), which is hereby incorporated by reference in its entirety).

If solely chemical shifts are considered, the unambiguous identification of peaks pairs is more involved whenever multiple peak pairs with degenerate chemical shifts in the other dimensions are present. The acquisition of the corresponding central peaks addresses this complication in a conceptually straightforward fashion. However, it is important to note that pairs of peaks generated by a chemical shift in-phase splitting have quite similar intensity. In contrast, peak pairs arising from different moieties, possible located in different amino acid residues, most often do not show similar intensity. This is because the nuclear spin relaxation times, which determine the peak intensities, vary within each residue as well as along the polypeptide chain. One may thus speak of a “nuclear spin relaxation time labeling” of peak pairs, which makes their identification an obvious task in most cases.

Using cryogenic probes can reduce NMR measurement times by about a factor of 10 or more (Flynn et al.,J. Am Chem. Soc.,122:4823–4824 (2000), which is hereby incorporated by reference in its entirety). Hence, the standard set of nine experiments (Table 2) could have been recorded with the same signal-to-noise ratios measured for the present study in about 6 hours using a cryogenic probe, i.e., the high sensitivity of cryogenic probes shifts even the recording of RD NMR experiments entirely into the sampling limited data acquisition regime. In view of this dramatic reduction in spectrometer time demand, minimally achievable RD NMR measurement times are of keen interest (Table 2) to be able to adapt the NMR measurement times to sensitivity requirements in future HTP endeavours.

If the standard set of experiments would have been recorded with a single transient per increment, 21.8 hours of spectrometer measurement time would have been required (Table 2). This is still about 3.5 times longer than the 6 hours alluded to above, which would be needed on a currently available cryogenic probe. To further reduce the measurement time, and in view of the aforementioned ‘spin relaxation time labeling’of peak pairs', one may then decide to also discard the use of13C-steady state magnetization for central peak detection. This would lead to a diminished requirement of 15.5 hours for the standard, or 8.1 hours for the minimal set of experiments (four projected 4D and two projected 3D spectra; Table 2). Hence, the measurement time of the minimal set of RD NMR experiments (which provides complete resonance assignments for Z-domain) could actually be neatly adjusted to the sensitivity requirements of a cryogenic probe.

Although RD NMR was proposed in 1993 (Szyperski et al.,J. Biomol. NMR,3:127–132 (1993); Szyperski et al.,J. Am. Chem. Soc.,115:9307–9308 (1993), which are hereby incorporated by reference in their entirety), its wide-spread use has been delayed by the more demanding spectral analysis when compared to conventional TR NMR. In particular, the necessity to extract chemical shifts from in-phase splittings suggests that strong computer support is key for employment of RD NMR on a routine basis. This can be readily addressed by using automated resonance assignment software for automated analysis of RD TR NMR data.

In conclusion, the joint employment of RD NMR spectroscopy, cryogenic probes, and automated backbone resonance assignment will allow one to determine a protein's backbone resonance assignments and secondary structure in a short time.

EXAMPLES

The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Sample Preparation

NMR measurements were performed using a 1 mM solution of uniformly13C/15N enriched “Z-domain” of theStaphylococcalprotein A (Tashiro et al.,J. Mol. Biol.,272:573–590 (1997); Lyons et al.,Biochemistry,32:7839–7845 (1993), which are hereby incorporated by reference in their entirety) dissolved in 90% D2O/10% H2O (20 mM K—PO4) at pH=6.5.

NMR Spectroscopy

Multidimensional NMR experiments (FIG. 1; Table 1) were recorded for a 1 mM solution of the 8.5 kDa protein “Z-domain” at a temperature of 25° C. The spectra (Table 2) were assigned, and the chemical shifts obtained from RD NMR (Table 3) were in very good agreement with those previously determined at 30° C. using conventional triple resonance (TR) NMR spectroscopy (Tashiro et al.,J. Mol. Biol.,272:573–590 (1997); Lyons et al.,Biochemistry,32:7839–7845 (1993), which are hereby incorporated by reference in their entirety).

NMR experiments were recorded at a temperature of 25° C. on a Varian Inova 600 spectrometer equipped with a new generation1H{13C,15N} triple resonance probe which exhibits a signal-to-noise ratio of 1200:1 for a standard 0.1% ethylbenzene sample. At 25° C., the correlation time for the overall rotational reorientation of the Z-domain was 4.5 ns (as inferred from measurements of T1ρ/T1polypeptide backbone15N spin relaxation time ratios (Kay et al.,Biochemistry,28:8972–8979 (1989); Szyperski et al.,J. Biomol. NMR,3:151–164 (1993), which are hereby incorporated by reference in its entirety)). This value was well within the 3–10 ns range usually encountered for medium-sized proteins at ambient temperatures. Hence, the results obtained in the framework of the present study were representative for medium-sized systems in the molecular weight range from about 5 to 20 kDa. NMR spectra were processed and analyzed using the programs PROSA (Güntert et al.,J. Biomol. NMR,2:619–629 (1992), which is hereby incorporated by reference in its entirety) and XEASY (Bartels et al.,J. Biomol. NMR,6:1–10 (1995), which is hereby incorporated by reference in its entirety), respectively.

Specific embodiments of the 8 new RD NMR experiments disclosed by the present invention as well as 3 other RD NMR experiments that have previously been published, were implemented for the present study.FIG. 1provides a survey of (i) the names, (ii) the magnetization transfer pathways and (iii) the peak patterns observed in the projected dimension of each of the 8 RD NMR experiments disclosed by the present invention as well as 3 other RD NMR experiments that have previously been published. The group comprising the first three experiments are designed to yield “sequential” connectivities via one-bond scalar couplings: 3DHα/βCα/β(CO)NHN (FIG. 1A; Szyperski et al.,J. Magn. Reson., B 105: 188–191 (1994), which is hereby incorporated by reference in its entirety), 3DHACA(CO)NHN (FIG. 1B), and 3DHC(C-TOCSY—CO)NHN (FIG. 1C). The following three experiments provide “intraresidual” connectivities via one-bond scalar couplings: 3D HNNCAHA(FIG. 1D; Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety), 3DHα/βCα/βCOHA (FIG. 1E), and 3DHα/βCα/βNHN (FIG. 1F). 3D HNN<CO,CA> (FIG. 1G; Szyperski et al.,J. Magn. Reson., B 108: 197–203 (1995); Szyperski et al.,J. Am. Chem. Soc.,118:8146–8147 (1996), which are hereby incorporated by reference in their entirety) offers both intraresidual1HN —13Cαand sequential1HN—13C′ connectivities. Although 3D HNNCAHA(FIG. 1D), 3DHα/βCα/βNHN (FIG. 1F) and 3D HNN<CO,CA> (FIG. 1G) also provide sequential connectivities via two-bond13Cαi−1—15Niscalar couplings, those are usually smaller than the one-bond couplings (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety), and obtaining complete backbone resonance assignments critically depends on experiments designed to provide sequential connectivities via one-bond couplings (FIGS. 1D–F). 3DHCCH—COSY (FIG. 1H) and 3DHCCH-TOCSY (FIG. 1I) allow one to obtain assignments for the “aliphatic” side chain spin systems, while 2DHBCB(CDCG)HD (FIG. 1J) and 2D1H-TOCSY-relayedHCH—COSY (FIG. 1K) provide the corresponding information for the “aromatic” spin systems.

The RD NMR experiments are grouped accordingly in Table 1, which lists for each experiment (i) the nuclei for which the chemical shifts are measured, (ii) if and how the central peaks are acquired and (iii) additional notable technical features. State-of-the art implementations (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996); Kay,J. Am. Chem. Soc.,115:2055–2057 (1993); Grzesiek et al.,J. Magn. Reson.,99:201–207 (1992); Montelione et al.,J. Am. Chem. Soc.,114:10974–10975 (1992); Boucher et al.,J. Biomol. NMR,2:631–637 (1992); Yamazaki et al.,J. Am. Chem. Soc.,115:11054–11055 (1993); Zerbe et al.,J. Biomol. NMR,7:99–106 (1996); Grzesiek et al.,J. Biomol. NMR,3:185–204 (1993), which are hereby incorporated by reference in their entirety) making use of pulsed field z-gradients for coherence selection and/or rejection, and sensitivity enhancement (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety) were chosen, which allow executing these experiments with a single transient per acquired free induction decay (FID). Semi (Grzesiek et al.,J. Biomol. NMR,3:185–204 (1993), which is hereby incorporated by reference in its entirety) constant-time (Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which is hereby incorporated by reference in its entirety) chemical shift frequency-labeling modules were used throughout in the indirect dimensions in order to minimize losses arising from transverse nuclear spin relaxation.FIGS. 2A–2Kprovide comprehensive descriptions of the RD NMR r.f. pulse sequences including eight previously unpublished RD NMR r.f. pulse schemes.

The maximal chemical shift evolution times, which largely determine the spectral resolution, as well as the measurement times invested for the present study (between 2.7 and 17.1 hours per spectrum) are given in Table 2. The S/N ratio achieved per unit of measurement time, i.e., the sensitivity, shows only little dependence on the relaxation delay between scans, Tdel, provided that 0.7·T1<Tdel<1.5·T1(Abragam,Principles of Nuclear Magnetism., Clarendon Press:Oxford (1986); Ernst et al.,Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford (1987), which are hereby incorporated by reference in their entirety). Hence, Trelwas set to rather short values around 0.7 seconds. Furthermore, to ensure efficient comparison of peak patterns and shapes manifested along the projected dimension in the various spectra, the RD NMR experiments in which1H and13C are jointly observed in the projected dimension (“HC”-type experiments;FIG. 1) were acquired with virtually the same maximal evolution time in t1(13C).

In total, fourteen RD TR NMR experiments were recorded: 3DHC(C-TOCSY—CO)NHN and 3DHCCH-TOCSY were acquired with two different mixing times (14 ms and 21 ms) each, and 3D HNNCAHAwere acquired with and without adiabatic decoupling of13Cβresonances for comparison (Kupce et al.,J. Magn. Reson., A 115:273–277 (1995); Matsuo et al.,J. Magn. Reson. B 113:190–194 (1996), which are hereby incorporated by reference in their entirety). Except for 3D HNNCAHA, 3D HNN<CO,CA> and 2D1H-TOCSY-relayedHCH—COSY (FIG. 1), central peaks were derived from13C magnetization (FIG. 1; Table 1). Hence, two subspectra, I and II containing the peak pairs and central peaks respectively, were generated (Szyperski et al.,J. Am. Chem. Soc.,118:8146–8147 (1996); Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which are hereby incorporated by reference in their entirety) for eight of the RD NMR experiments (FIG. 1). Overall, twenty-four processed RD NMR (sub)spectra were thus obtained for a detailed exploration of relative sensitivities and data collection strategies. These were complemented with conventional 3D HNNCACB data (Table 2; Wittekind et al.,J. Magn. Reson., B 101:201–205 (1993), which is hereby incorporated by reference in its entirety).

Adjustment of r.f Carrier Frequencies to Minimize Spectral Overlap

In view of potential peak overlap in spectra recorded for larger proteins, it is of central importance to properly set the r.f. carrier frequencies. An illustrative example is the 3D HNNCAHAexperiment, where adjustments of the carrier frequencies allows one to place central peaks and upfield and downfield component of the peak pairs into three separated spectral regions (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety). This is accomplished by choosing a1H-carrier frequency that yields a minimal in-phase splitting exceeding the13Cαchemical shift dispersion (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety). As a consequence, the generation of peak pairs does not lead to increased spectral overlap. In fact, the increase in the number of peaks expected for 3D HNNCAHArelative to 3D HNNCA was comparable to the increase observed in widely used conventional 3D HNNCACB. 3D HNNCACB exhibited up to four peaks for each amino acid residue: (Wittekind et al.,J. Magn. Reson., B 101:201–205 (1993), which is hereby incorporated by reference in its entirety) an intraresidue and a sequential connectivity in each of the quite well separated spectral regions containing the13Cαand13Cβresonances, respectively. Similarly, 3D HNNCAHAcomprised the three separated regions each of which may exhibit one intraresidual and one sequential connectivity per amino acid residue (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety).

Sensitivity Analysis of RD NMR Experiments

Since a reduction of dimensionality in a NMR experiment preserves the relative sensitivity of the higher-dimensional parent experiments, evaluating the relative sensitivity of an entire set of multidimensional NMR experiments designed to provide complete resonance assignment for a protein is of general interest. The relative sensitivity of the RD NMR and 3D HNNCACB experiments were analyzed first, by determining the yield of peak detection, i.e., the ratio of observed peaks over the total number of expected peaks, and second, by separately assessing the S/N ratio distributions of peaks belonging to either RD peak pairs or central peaks. Moreover, distinct S/N distributions were then generated according to (i) the atom position involved (e.g., α- or β-moiety inHα/βCα/β(CO)NHN), (ii) the involvement of intraresidue or sequential connectivities (e.g.,13Cαi—1HNiand13Cαi−1—1HNiconnectivities inHα/βCα/βNHN) and (iii) the classification of COSY-type, relay and double-relay peaks inHCCH TOCSY. In total, 127 S/N distributions were thus analyzed (FIG. 5; Table 4). For 3DHα/βCα/β(CO)NHN (FIG. 1A) and 3DHα/βCα/βCOHA (FIG. 1E), there were 4 distributions each: α- and β-connectivities in subspectra I and II. For 3DHACA(CO)NHN (FIG. 1B) and 2DHBCB(CDCG)HD (FIG. 1J), there were 2 distributions each: connectivities in subspectra I and II. For 3DHC(C-TOCSY—CO)NHN (FIG. 1C) recorded with 14 and 21 ms mixing time, respectively, there were 10 distributions each: α-, β-, γ-, δ- and ε-connectivities in subspectra I and II. For 3D HNNCAHA(FIG. 1D), there were 8 distributions: intraresidual and sequential connectivities recorded with and without adiabatic13Cβdecoupling. For 3DHα/βCα/βNHN (FIG. 1F), there were 8 distributions: intraresidual and sequential α- and β-connectivities in subspectra I and II. For 3D HNNCACB, there were 4 distributions: intraresidual and sequential α- and β-connectivities. For 3D HNN<CO,CA> (FIG. 1G), there were 2 distributions: peak pairs and central peaks. For 3DHCCH—COSY (FIG. 1H), there were 10 distributions: connectivities detected on α-, β-, γ-, δ- and ε-protons for subspectra I and II. For 3DHCCH-TOCSY (FIG. 1H) recorded with 14 and 21 ms mixing time, there were 30 distributions each: COSY-type, relay and double-relay connectivities detected on (α-, β-, γ-, δ- and ε-protons for subspectra I and II. For 2D1H-TOCSY-relayedHCH—COSY (FIG. 1K), there were 3 distributions for connectivities detected on δ-, ε- and ζ-protons. In order to exclude a bias arising from longer transverse relaxation times in several highly disordered terminal residues (Tashiro et al.,J. Mol. Biol.,272:573–590 (1997); Lyons et al.,Biochemistry,32:7839–7845 (1993), which are hereby incorporated by reference in their entirety), the N-terminal octapeptide segment comprising residues “−13” to “−6” (in the numbering chosen in Tashiro et al.,J. Mol. Biol.,272:573–590 (1997) and Lyons et al.,Biochemistry,32:7839–7845 (1993), which are hereby incorporated by reference in their entirety) was not considered for the current sensitivity analyses. To rank the NMR experiments (Table 2) according to relative sensitivity, focus was put on (i) the peak detection yield and (ii) the averaged S/N ratios of those peak categories encoding the prime information to be obtained from a given spectrum, i.e., intraresidual connectivities in HNNCAHA(FIG. 1D),Hα/βCα/βCOHA (FIG. 1E),Hα/βCα/βNHN (FIG. 1F) and HNNCACB, correlation peaks in HCCH—COSY and relay connectivities inHCCH TOCSY. For comparison, these averaged S/N ratios were subsequently divided by the square-root of the NMR measurement time (Tables 2 and 4) and scaled relative to the most sensitive experiment, i.e.,HACA(CO)NHN (Table 4;FIG. 5).

In principle, the relative sensitivities of NMR experiments can be estimated by calculating transfer amplitudes (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998); Ernst et al.,Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford (1987); Wittekind et al.,J. Magn. Reson., B 101:201–205 (1993); Buchler et al.,J. Magn. Reson.,125:34–42 (1997), which are hereby incorporated by reference in their entirety). However, these calculations rely on various assumptions such as knowledge about nuclear spin relaxation times, or neglect of B1-inhomogeneity and imperfections of composite pulse decoupling sequences. Hence, an experimental approach is mandatory to obtain a thorough sensitivity assessment, in particular for the experiments employed for side chain resonance assignments.

The key yields of peak detection as well as the relative sensitivity of the NMR spectra recorded for the present study (Tables 1 and 2) are shown inFIG. 5. The S/N distribution analysis that was required to generateFIG. 5is provided in Table 4. Since adiabatic13Cβdecoupling (Kupce et al.,J. Magn. Reson., A115:273–277 (1995); Matsuo et al.,J. Magn. Reson. B 113:190–194(1996), which are hereby incorporated by reference in their entirety) increased the sensitivity of 3D HNNCAHAby a factor of about 1.5 (FIG. 5; Table 4), only the decoupled spectrum was considered in this analysis. Among the group of experiments designed to yield sequential connectivities (FIG. 4), all of the expected peaks were detected for 3DHACA(CO)NHN (FIGS. 1B and 4) and 3DHα/βCα/β(CO)NHN (FIGS. 1A and 4). In spite of the rather long measurement time of 17 hours (Table 2), a substantial fraction of the expected cross peaks was not observed for 3DHC(C-TOCSY—CO)NHN (FIGS. 1C and 4). Evidently, losses due to rotating frame transverse relaxation and off-resonance effects during the C—C TOCSY relay are significantly larger than those encountered when implementing the C—C COSY step which expands 3DHACA(CO)NHN to 3DHα/βCα/β(CO)NHN. Moreover, due to the oscillatory nature of the spin modes associated with total correlation, (Ernst et al.,Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford (1987), which is hereby incorporated by reference in its entirety) the average S/N ratio observed for a given atom position critically depends on the particular choice of the mixing time in 3DHC(C-TOCSY—CO)NHN (Table 4): e.g., several β-moiety signals are lost at the expense of detecting additional γ-, δ- or ε-moiety cross peaks for the long aliphatic side chains when increasing the mixing time from 14 ms to 21 ms (FIG. 4).

Among the experiments providing intraresidue connectivities (FIG. 6), HNNCAHA(FIGS. 1D and 6A) exhibited complete detection of expected peaks and a sensitivity which is comparable toHα/βCα/β(CO)NHN, but significantly higher thanHα/βCα/βCOHA (FIGS. 1E and 6B) andHα/βCα/βNHN (FIGS. 1F and 6C). The latter experiment, designed in an ‘out-and-stay fashion’ as CBCANHN (Kay,J. Am. Chem. Soc.,115:2055–2057 (1993), which is hereby incorporated by reference in its entirety), is the least sensitive among the suite of RD NMR experiments studied here and can thus be expected to be primarily of use for smaller proteins. However, virtually all expected correlations were observed. Conventional HNNCACB is slightly more sensitive than HNNCAHAand equally sensitive asHα/βCα/β(CO)NHN. However, when considering symmetrization of [ω1(13C),ω3(1HN)]-strips about central peaks along ω1, (Szyperski et al.,J. Magn. Reson., B 108: 197–203 (1995); Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which are hereby incorporated by reference in their entirety) HNNCAHAcan be considered to be more sensitive than HNNCACB even for smaller proteins. HNN<CO,CA> (FIG. 1G) offers both intraresidue1HN—13Cα(peak pairs) and sequential1HN—13C′ (central peaks) connectivities. In accordance with the outstanding sensitivity of HNNCO, central peak detection in HNN<CO,CA> was by far the most sensitive observed in all spectra, while the sensitivity of corresponding peak pair detection was comparable to HNNCAHA. Hence, central peaks in 3D HNN<CO,CA> may be recruited for secure spin system identification (Zimmerman et al.,J. Mol. Biol.,269:592–610 (1997), which is hereby incorporated by reference in its entirety) in cases of overlap in 2D [15N,1H]—HSQC.

The sensitivity of peak pair detection in 3DHCCH COSY, required for aliphatic side chain assignment, was again comparable to 3D HNNCAHA, while detection of relayed COSY peaks in 3DHCCH TOCSY was slightly less sensitive. The incompleteness of relay peak detection was, however, to some extent due to signal overlap (Table 4). 2DHBCB(CDCG)HD and 2D1H-TOCSY-relayedHCH—COSY, providing the aromatic spin system assignments, appeared to be rather sensitive. However, analysis for the Z-domain was biased by (i) the relatively small number of aromatic residues, and (ii) their partly flexibly disordered nature (His(−4), Phe 5 and Phe 13 exhibit local displacements that are well above the average for residues buried in the molecular core; protein data bank accession code: 2SPZ). When involving only those aromatic rings that are apparently not flexibly disordered, 2DHBCB(CDCG)HD appeared to be slightly less sensitive than 3DHCCH COSY.

Overall (FIG. 5), (i) outstanding sensitivity was found for 3DHACA(CO)NHN, (ii) similar sensitivity was found for 3DHα/βCα/β(CO)NHN, 3D, HNNCAHA, 3D HNN<CO,CA>, 3D HNNCACB, 3DHCCH COSY and 2D1H-TOCSY-relatedHCH—COSY, (iii) slightly reduced sensitivity was found for 3DHα/βCα/βCOHA, 2DHBCB(CDCG)HD and relay peak detection in 3DHCCH TOCSY, and (iv) the lowest sensitivity was found for 3DHC(C-TOCSY—CO)NHN and 3DHα/βCα/βNHN. In the “Hα/βCα/β-experiments, the averaged intensity of the α- and β-moiety peak pairs was quite similar (though the S/N distribution of the β-peaks was broader reflecting larger variations in transverse relaxation times), and the central peaks exhibited a sensitivity of about two thirds relative to the individual peaks of the peak pairs. However, since the non-selective13C T1-relaxation times are shorter than the1H T1-times at higher molecular weight (Abragam,Principles of Nuclear Magnetism., Clarendon Press:Oxford (1986); Ernst et al.,Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press:Oxford (1987), which are hereby incorporated by reference in their entirety), the relative sensitivity of central peak detection using13C-magnetization becomes more favorable for larger systems. Moreover, the relative sensitivity of the various experiments shifts relative to each other with increasing molecular weight (Buchler et al.,J. Magn. Reson.,125:34–42 (1997), which is hereby incorporated by reference in its entirety). In particular, 3D HNNCACB and 3DHα/βCα/βCOHA can be expected to loose relative sensitivity for larger systems since transverse magnetization resides comparably long on rapidly relaxing13Cα.

HTP Assignment Strategy: A “Standard Set” of RD NMR Experiments

The comprehensive analysis of the suite of multidimensional spectra recorded for the present study (FIG. 5; Tables 1 and 2) lays the foundation to devise strategies for RD NMR-based HTP resonance assignment of proteins.

For proteins in the molecular weight range up to about 20 kDa, 3DHα/βCα/β(CO)NHN plays a pivotal role (FIG. 7). Firstly, the peak patterns observed along ω1(13Cα/β) in subspectra I and II enable sequential resonance assignment in combination with HNNCAHAand HNNCACB, respectively, by matching intraresidue and sequential1Hα,13Cαand13Cβchemical shifts (FIG. 8). (When considering ‘nuclear spin relaxation time labeling’ of peak pairs, subspectrum II derived from13C steady state magnetization provides largely redundant information when compared with subspectrum I. However, the observation of the central peaks allows direct matching of peak positions between subspectrum II, essentially a CBCA(CO)NHN spectrum, and HNNCACB (FIG. 6).) Moreover, this set of chemical shifts alone provides valuable information for amino acid type identification (Zimmerman et al.,J. Mol. Biol.,269:592–610 (1997); Cavanagh et al.,Protein NMR Spectroscopy, Academic Press, San Diego, (1996); Grzesiek et al.,J. Biomol. NMR,3:185–204 (1993), which are hereby incorporated by reference in their entirety). Complementary recording of 3DHα/βCα/βCOHA and 3D HNN<CO,CA> contributes polypeptide backbone13C═O chemical shift measurements for establishing sequential assignments: the intraresidue correlation is obtained by ω1(13Cα/β) peak pattern matching (FIGS. 9A–B) with 3DHα/βCα/β(CO)NHN, and the sequential correlation is inferred from13Cα,15N and1HNchemical shifts in 3D HNN<CO,CA> (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety). Notably, even for medium-sized (non-deuterated) proteins this approach is superior to the use of a low sensitivity HNNCACO-type experiment (e.g., in combination with HNNCOCA), where the magnetization transfer via rapidly relaxing13Cαrelies on the rather small15N—13Cαone-bond scalar coupling. Secondly, comparison of ω1(13Cα/β) peak patterns with 3DHCCH—COSY (FIG. 10) and TOCSY connects the Cα/β/Hα/βchemical shifts with those of the aliphatic side chain spin systems (For Z-domain, complete side chain assignments were obtained for all but six residues using 3DHCCH—COSY only.) (FIGS. 10 and 11), while comparison of ω1(13Cβ) peaks with 2DHBCB(CDCG)HD and subsequent linking with1Hδchemical shifts detected in 2D1H-TOCSY-relayedHCH—COSY affords assignment of the aromatic spin systems (FIG. 12). Since for many amino acid residues the two β-protons exhibit non-degenerate chemical shifts, the connection ofHα/βCα/β(CO)NHN andHBCB(CDCG)HD orHCCH—COSY/TOCSY (FIG. 7) may in fact often rely on comparison of three chemical shifts, i.e., δ(1Hβ2), δ(1Hβ3) and δ(13Cβ). This consideration underscores the potential of recruiting β-proton chemical shifts for establishing sequential resonance assignments.

The ‘standard set’ of nine experiments (labeled with asterisks in Table 2) as described in the above paragraph required 60 hours of instrument time for the Z-domain on our 600 MHz NMR system (Table 2). However, the minimal S/N ratios detected (Table 4) reveal that half of the measurement time would have been sufficient for backbone amide proton detected experiments, indicating that these spectra were still acquired in the sampling limited regime. (The lowest S/N peak ratios are around 5:1, which implies that a reduction of by could be afforded. A further indication of an inappropriately long measurement time is due to the fact that nearly all sequential connectivities relying on two-bond scalar couplings (Güntert et al.,J. Biomol. NMR,2:619–629 (1992), which is hereby incorporated by reference in its entirety) were observed in 3D HNNCAHA(FIG. 7): nearly all1HN,15N,13Cαand1Hαbackbone resonances of Z-domain could be assigned using this spectrum (Szyperski et al.,J. Biomol. NMR,11:387–405 (1998), which is hereby incorporated by reference in its entirety) alone.) Hence, a nearly complete resonance assignment of the Z-domain could have been obtained from the standard set in about 40 hours, if the RD backbone experiments were conducted with a single transient per acquired FID. (The suite of experiments in Table 1 may provide complete resonance assignments of proteins, excluding only the side chain NHnmoieties, the CHεgroups of histidinyl, and the CHε3, CHζ2,3and CHη2groups of tryptophanyl residues, which can be obtained as described in Yamazaki et al.,J. Am. Chem. Soc.,115:11054–11055 (1993), which is hereby incorporated by reference in its entirety. Notably, the protein studied here does not contain tryptophan residues.) This outstandingly short measurement time needs to be compared with 1–3 weeks of measurement time that are currently routinely invested to assign medium-sized proteins. Concomitantly, the high redundancy for establishing sequential connectivities using this suite of experiments (six projected 4D, one 3D and two projected 3D experiments) greatly supports robust automated assignment. Importantly, the information encoded in each projected 4D spectrum cannot be obtained by simply recording two 3D spectra: in cases of chemical shift degeneracy a chemical shift quartuple is not equivalent to two shift triples.

Sensitivity Profile Within the “Standard Set” of NMR Experiments

It is desirable that the NMR experiments applied for protein resonance assignment in a high-throughput manner exhibit comparable sensitivity. This is because the prediction of the totally required measurement times is facilitated (roughly a multiple of the measurement time of an arbitrarily chosen single experiment) and the signal-to-noise ratios observed in the experiment conducted first allow one to readily adjust the (rather similar) measurement times of the remaining ones while the recording of the set of experiments is in progress. It is thus important to note that the sensitivity within the standard set of nine experiments (Table 2) varies by only about a factor of two when comparing peak pair detection in 3DHα/βCα/β(CO)NHN with relay COSY peak detection in 3DHCCH-TOCSY (FIG. 5). Extraordinarily sensitive central peak detection in 3D HNN<CO,CA> represents the sole exception. However, the availability of extremely sensitive detection of (1HN,15N,13C═O) chemical shift triples is of high value for identification of spin systems (Zimmerman et al.,J. Mol. Biol.,269:592–610 (1997), which is hereby incorporated by reference in its entirety). In fact, this apparent exception thus neatly complements the even sensitivity profile of the remaining experiments.

A “Minimal Set” of RD NMR Experiments

For Z-domain, six RD NMR experiments were actually sufficient to provide the desired resonance assignments: 3DHα/βCα/β(CO)NHN, 3D HNNCAHA, 3DHCCH—COSY/TOCSY, 2DHBCB(CDCG)HD and 2D1H-TOCSY-relayedHCH—COSY. This set of experiments was recorded within 36 hours of instrument time (Table 2), and can be considered as a ‘minimal set’ of RD NMR experiments for HTP resonance assignment of proteins up to around 10 kDa. For smaller proteins, the use of 3DHC(C-TOCSY—CO)NHN, 3DHα/βCα/βNHN, 3DHCCH—COSY, 2DHBCB(CDCG)HD and 2D1H-TOCSY-relayedHCH—COSY represents a viable alternative to rapidly obtain assignments (Table 1).