bluuebunny/crossref_metadata_2025 huggingface dataset:
Backbone dynamics of the human cc chemokine eotaxin: Fast motions, slow motions, and implications for receptor binding
Authors: Crump Matthew P. , Spyracopoulos Leo , Lavigne Pierre , Kim Key‐Sun , Clark‐Lewis Ian , Sykes Brian D.
Published: February 2009

Eotaxin is a member of the chemokine family of about 40 proteins that induce cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is highly expressed by eosinophils, and it is considered important in the pathology of chronic respiratory disorders such as asthma. The high resolution structure of eotaxin is known. The 74 amino acid protein has two disulfide bridges and shows a typical chemokine fold comprised of a core of three antiparallel β‐strands and an overlying α‐helix. In this paper, we report the backbone dynamics of eotaxin determined through 15N‐T1, T2, and [H]‐15N nuclear Overhauser effect heteronuclear multidimensional NMR experiments. This is the first extensive study of the dynamics of a chemokine derived from 600, 500, and 300 MHz NMR field strengths. From the T1, T2, and NOE relaxation data, parameters that describe the internal motions of eotaxin were derived using the Lipari‐Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure elements. However, surrounding the core, the regions known to be functionally important in chemokines show a range of motions on varying timescales. These include extensive subnanosecond to picosecond motions in the N‐terminus, C‐terminus, and the N‐loop succeeding the disulfides. Analysis of rotational diffusion anisotropy of eotaxin and chemical exchange terms at multiple fields also allowed the confident identification of slow conformational exchange through the “30s” loop, disulfides, and adjacent residues. In addition, we show that these motions may be attenuated in the dimeric form of a synthetic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.
Probing the conformation of a human apolipoprotein C‐1 by amino acid substitutions and trimethylamine‐N‐oxide
Authors: Gursky Olga
Published: February 2009

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine‐N‐oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C‐1 (apoC‐1, 57 residues) containing point mutations in the predicted α‐helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild‐type and plasma apoC‐1 in neutral low‐salt solutions superimpose, indicating 31 ± 4% α‐helical structure at 22 °C that melts reversibly with Tm,WT = 50 ± 2°C and van't Hoff enthalpy ΔHv,WT(Tm) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α‐helical content of 42 ± 4% and an increased Tm,G15A = 57 ± 2°C, which corresponds to stabilization by ΔGapp = +0.4 ± 1.5 kcal/mol. G15P mutant has ∼∼20% α‐helical content at 22 °C and unfolds with low cooperativity upon heating to 90 °C. R23P and T45P mutants are fully unfolded at 0–90 °C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative α‐helical unit in apoC‐1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self‐associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil‐to‐helix transition in apoC‐1, with the maximal α‐helical content reaching 74%. Comparison with the maximal α‐helical content of 73% observed in lipid‐bound apoC‐1 suggests that the TMAO‐stabilized secondary structure resembles the functional lipid‐bound apolipoprotein conformation.
New evidence for the denaturant binding model
Authors: Wu Jia‐Wei , Wang Zhi‐Xin
Published: February 2009

Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.
Global and local dynamics of the human U1A protein determined by tryptophan fluorescence
Authors: Jean John M. , Clerte Caroline , Hall Kathleen B.
Published: February 2009

Tryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N‐terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1‐101; the C‐terminal RBD2, residues 202‐282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the β‐sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady‐state and time‐resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the β‐sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.
Position dependence of amino acid intrinsic helical propensities II: Non‐charged polar residues: Ser, Thr, Asn, and Gln
Authors: Petukhov Michael , Uegaki Koichi , Yumoto Noboru , Yoshikawa Susumu , Serrano Luis
Published: February 2009

The assumption that the intrinsic α‐helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine‐based peptide. We found that Thr is more favorable (by ∼0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (±0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (±0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.
Conformational changes in the activation loop of the insulin receptor's kinase domain
Authors: Frankel Mark , Bishop Steven M. , Ablooglu Ararat J. , Han Yuan‐Ping , Kohanski Ronald A.
Published: February 2009

Low catalytic efficiency of basal‐state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo‐IRKD. Bound Mg‐adenine nucleoside di‐ and triphosphates increased the cleavage rate with half‐maximal effects observed at 0.4‐0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino‐terminal and carboxy‐terminal core‐flanking regions of the IRKD had no statistically significant impact on the ligand‐dependent or ‐independent activation loop cleavages. Furthermore, the core‐flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride‐induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses ≥90% of the ligand‐free basal state kinase. However, normal intracellular concentrations of Mg‐adenine nucleotides, which are in the millimolar range, would favor a basal‐state conformation of the activation loop that is more accessible.
Design of highly stable functional GroEL minichaperones
Authors: Wang Qinghua , Buckle Ashley M. , Foster Nicholas W. , Johnson Christopher M. , Fersht Alan R.
Published: February 2009

GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193‐345). A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations. Two small‐to‐large mutations, A223T, A223V and one similar‐size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol−1 each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol−1 more stable than wild‐type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.
Crystal structure of the EF‐hand parvalbumin at atomic resolution (0.91 Å) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core
Authors: Declercq Jean‐Paul , Evrard Christine , Lamzin Victor , Parello Joseph
Published: February 2009

Several crystal structures of parvalbumin (Parv), a typical EF‐hand protein, have been reported so far for different species with the best resolution achieving 1.5 Å. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+‐loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 Å). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three‐dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well‐defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 Å. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy‐barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time‐dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.
The interaction of neurotrophins with the p75NTR common neurotrophin receptor: A comprehensive molecular modeling study
Authors: Shamovsky Igor L. , Weaver Donald F. , Ross Gregory M. , Riopelle Richard J.
Published: February 2009

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5), to p75NTR is studied by molecular modeling based on X‐ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine‐rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT‐3 and NT‐4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.
The turn sequence directs β‐ strand alignment in designed β‐hairpins
Authors: Alba Eva De , Rico Manuel , Jiménez M. Angeles
Published: February 2009

A previous NMR investigation of model decapeptides with identical β‐strand sequences and different turn sequences demonstrated that, in these peptide systems, the turn residues played a more predominant role in defining the type of β‐hairpin adopted than cross‐strand side‐chain interactions. This result needed to be tested in longer β‐hairpin forming peptides, containing more potentially stabilizing cross‐strand hydrogen bonds and side‐chain interactions that might counterbalance the influence of the turn sequence. In that direction, we report here on the design and 1H NMR conformational study of three β‐hairpin forming pentadecapeptides. The design consists of adding two and three residues at the N‐ and C‐termini, respectively, of the previously studied decapeptides. One of the designed pentadecapeptides includes a potentially stabilizing R‐E salt bridge to investigate the influence of this interaction on β‐hairpin stability. We suggest that this peptide self‐associates by forming intermolecular salt bridges. The other two pentadecapeptides behave as monomers. A conformational analysis of their 1H NMR spectra reveals that they adopt different types of β‐hairpin structure despite having identical strand sequences. Hence, the β‐turn sequence drives β‐hairpin formation in the investigated pentadecapeptides that adopt β‐hairpins that are longer than the average protein β‐hairpins. These results reinforce our previous suggestion concerning the key role played by the turn sequence in directing the kind of β‐hairpin formed by designed peptides.
Intrabody construction and expression III: Engineering hyperstable VH domains
Authors: Wirtz Peter , Steipe Boris
Published: February 2009

The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin VL domains. Here we apply rational stability engineering by consensus sequence analysis to VH domains. Isolated VH domains tend to aggregate more easily than VL domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the VH domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20‐fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated VH domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the VL domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.
Folding of an isolated ribonuclease H core fragment
Authors: Chamberlain Aaron K. , Fischer Kael F. , Reardon Deirdre , Handel Tracy M. , Marqusee Susan
Published: February 2009

Based on results from both equilibrium and kinetic hydrogen exchange studies of Escherichia coli ribonuclease HI (RNase H), a fragment of RNase H (eABCD) was designed. The sequence of eABCD contains less than half of the protein's primary sequence and includes the regions that were shown to be the most protected from hydrogen exchange in all previous studies of RNase H. This core fragment of RNase H encodes a well‐ordered protein with native‐like properties. When isolated from the full‐length monomeric protein, the eABCD fragment forms a stable dimer. However, we show indirectly that the monomeric form of eABCD is folded and has an overall secondary structure similar to the dimeric form.
Crystal structure of brain‐type creatine kinase at 1.41 Å resolution
Authors: Eder Michael , Schlattner Uwe , Wallimann Theo , Becker Andreas , Kabsch Wolfgang , Fritz‐Wolf Karin
Published: February 2009

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue–as well as compartment‐specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age‐related diseases. The crystal structure of chicken cytosolic brain‐type creatine kinase (BB‐CK) has been solved to 1.41 Å resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N‐terminal region (2‐12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+‐mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N‐terminal conformation and secondary structure.

The high‐resolution structure of BB‐CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform‐specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB‐CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N‐terminal domain by rms 4 Å from the structure without substrates.
Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly
Authors: Schneider Gunter , Sandalova Tatyana , Persson Karina , Jordan Douglas B. , Viitanen Paul V.
Published: February 2009

Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60‐meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5‐nitroso‐6‐ribitylamino‐2,4‐pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 Å resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four‐stranded parallel β‐sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N‐terminal β‐strand interacts with the β‐sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand β1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the b‐hairpin connecting helices α4 and α5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface.
Crystal structure of reduced thioredoxin reductase from Escherichia coli: Structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor
Authors: Lennon Brett W. , Williams Charles H. , Ludwig Martha L.
Published: February 2009

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X‐ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800‐816). This form of TrxR, denoted FO, permits the reaction of enzyme‐bound reduced FAD with a redox‐active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X‐ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34° “butterfly” bend about the N(5)‐N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15‐28° for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side‐chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.
Production of soluble αβ T‐cell receptor heterodimers suitable for biophysical analysis of ligand binding
Authors: Willcoj Benjamin E. , Gao George F. , Wyer Jessica R. , O'callaghan Christopher A. , Jakobsen Bent K. , Boulter Jonathan M. , Bell John I. , Jones E. Yvonne , Van Der Merwe P. Anton
Published: February 2009

A method to produce αβ T‐cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c‐Jun (alpha) or v‐Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA‐A2‐restricted matrix epitope (M58‐66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody‐reactive, and stable at concentrations lower than 1 μM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366‐374) restricted by H2‐Db. Production of these receptors has facilitated a detailed analysis of viral peptide‐Major Histocompatibility Complex (peptide‐MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR‐peptide‐MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.
Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4‐thiouridine biosynthesis
Authors: Mueller Eugene G. , Palenchar Peter M.
Published: February 2009

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4‐thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique “P‐loop” motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4‐thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site‐directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4‐thiouridine.
Thermodynamics of replacing an α‐helical Pro residue in the P40S mutant of Escherichia coli thioredoxin
Authors: Chakrabarti Atis , Srivastava Sarika , Swaminathan Chittoor P. , Surolia Avadhesha , Varadarajan Raghavan
Published: February 2009

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long α‐helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Sen The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L, P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L, P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy‐entropy compensation was observed and the two proteins had similar values of ΔCp. Thus, replacement of a Pro in the interior of an α helix can have substantial effects on protein stability.
Structures of yeast vesicle trafficking proteins
Authors: Tishgarten Tomer , Yin Fang Fang , Grant Tracy R. , Lipscomb Leigh Ann , Faucher Keith M. , Dluhy Richard A. , Stevens Tom H. , Mollard Gabriele Fischer Von
Published: February 2009

In protein transport between organelles, interactions of v‐ and t‐SNARE proteins are required for fusion of proteincontaining vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q‐SNAREs) or arginine (R‐SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v‐ and t‐SNARE proteins, Vti 1p and Pep12p, that are Q‐SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly α‐helical and that Vti 1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R‐SNARE proteins characterized previously. The α‐helicity of Vti1p is similar to that of Q‐SNARE proteins characterized previously. Pep12p, a Q‐SNARE, is highly α‐helical. It is distinct from other Q‐SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.
A common mechanism for recombinant human NGF, BDNF, NT‐3, and murine NGF slow unfolding
Authors: De Young Linda R. , Schmelzer Charles H. , Burton Louis E.
Published: February 2009

The recombinant human nerve growth factor (hNGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed‐phase high‐performance liquid chromatography, and sizeexclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half‐lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT‐3, and BDNF, respectively, at 25 °C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT‐3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT‐3 the slowest to refold and BDNF the fastest. Threading of the N‐terminus out through the cystine‐knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine‐knot loop (14 for hNGF, mNGF, NT‐3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT‐3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.
The effects of alpha‐helix on the stability of Asn residues: Deamidation rates in peptides of varying helicity
Authors: Kosky Andrew A. , Razzaq Ursula O. , Treuheit Michael J. , Brems David N.
Published: February 2009

Asn deamidation was monitored in Ala‐based octadecapeptides of varying α‐helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala → Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed‐phase high‐performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of α‐helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.
Molecular basis for triclosan activity involves a flipping loop in the active site
Authors: Qiu Xiayang , Abdel‐Meguid Sherin S. , Janson Cheryl A. , Court Robert I. , Smyth Martin G. , Payne David J.
Published: February 2009

The crystal structure of the Escherichia coli enoyl reductase‐NAD +‐triclosan complex has been determined at 2.5 Å resolution. The Ile192‐Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure‐based inhibitor design.
Thermolysin and mitochondrial processing peptidase: How far structure—functional convergence goes
Authors: Grishin Nick V. , Makarova Kira S.
Published: February 2009

The structure—functional convergence between two Zndependent proteases, namely thermolysin and mitochondrial processing peptidase (MPP), is described. These two families of nonhomologous enzymes show not only functional convergence of several active site residues as in chymotrypsin and subtilisin, but also structural convergence of overall molecular architectures including the β‐sheet arrangement and packing of the surrounding α‐helices. The major functionally important structural elements are present in both enzymes with different topological connections and often in reverse main‐chain orientation, but display similar packing. The structural comparison helps to rationalize sequence “inversion” of the HEXXH thermolysin consensus present as HXXEH in MPP. The described structural convergence may be due to a limited number of alternatives to build a Zn‐protease that utilizes hydrogen bonding between a substrate main chain and the enzyme β‐sheet for substrate binding.
Changing the surface of a virus shell fusion of an enzyme to polyoma VP1
Authors: Gleiter Stefan , Stubenrauch Kay , Lilie Hauke
Published: February 2009

Recent developments on virus‐like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell‐type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild‐type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased KM values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties.
Structure of the human glycogen‐associated protein phosphatase 1 regulatory subunit hGM: Homology modeling revealed an (α/β)8‐barrel‐like fold in the multidomain protein
Authors: Souchet Michel , Legave Marie‐Noelle , Bril Antoine , Berrebi‐Bertrand Isabelle , Jullian Nathalie , Bertrand Hugues‐Olivier
Published: February 2009

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (α/β)8‐barrel‐like structure within the N‐ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three‐dimensional (3D) model for the 381 N‐ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology‐derived model includes the putative glycogen binding area located within the 142–230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51–67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X‐ray studies (Egloff et al., 1997). Finite difference Poisson‐Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.
The structures of the neurotrophin 4 homodimer and the brain‐derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk‐binding site
Authors: Robinson Robert C. , Choe Senyon , Radziejewski Czeslaw , Spraggon Glen , Greenwald Jason , Kostura Mikey R. , Burtnick Leslie D. , Stuart David I. , Jones E. Yvonne
Published: February 2009

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain‐derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.
α1‐Microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound
Authors: Berggård Tord , Lindqvist Annika , Cedervall Tommy , Åkerström BO , Thøgersen Ida B. , Enghild Jan J. , Cohen Arieh , Persson Per , Jönsson Jan‐Åke , Silow Maria
Published: February 2009

α1‐Microglobulin (α1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three‐dimensional (3D) structure that forms an internal hydrophobic ligand‐binding pocket, α1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty‐one different colored tryptic peptides corresponding to residues 88–94, 118–121, and 122–134 of human α1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of α1m. A 3D model of α1m, based on the structure of rat epididymal retinoic acid‐binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of α1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of α1m is complex bound to IgA. Only the free α1m carried colored groups, whereas α1m linked to IgA was uncolored.
Three‐dimensional solution structure of mouse [Cd7]‐metallothionein‐l by homonuclear and heteronuclear NMR spectroscopy
Authors: Zangger Klaus , ÖZ GÜLIN , Armitage Ian M. , Otvos James D.
Published: February 2009

Sequential 1H‐NMR assignments of mouse [Cd7]‐metallothionein‐l (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion‐heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three‐dimensional structure was then calculated using the distance constraints from two‐dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys‐Cd connectivities as input for a distance geometry‐dynamical simulated annealing protocol in X‐PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N‐terminal β‐ and the C‐terminal α‐domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine‐metal connectivities. The only elements of regular secondary structure found were two short stretches of 310 helices along with some half‐turns in the α‐domain. Structural comparison with rat liver MT2 showed high similarity, with the β‐domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium‐cadmium exchange rates found in the β‐domain of MT1.
Methionine‐141 directly influences the binding of 4‐methylpyrazole in human σσ alcohol dehydrogenase
Authors: Xie Peiguang T. , Hurley Thomas D.
Published: February 2009

Pyrazole and its 4‐alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human σσ isoenzyme exhibits a 580‐fold lower affinity for 4‐methylpyrazole than does the human β1β1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4‐methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human σσ alcohol dehydrogenase (ADH) isoenzyme to 2.5 Å resolution. Comparison of this structure to the human β1β1 isoenzyme structure indicated that the side‐chain position for Met141 in σσ ADH might interfere with 4‐methylpyrazole binding. Mutation of Met141 in σσ ADH to Leu (σσ141L) lowers the Ki for 4‐methylpyrazole from 350 to 10 μM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate‐binding pocket at a position close to the methyl group of 4‐methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X‐ray structure of the σ141L mutant enzyme was determined to 2.4 Å resolution. The three‐dimensional structure of the mutant enzyme is identical to the wild‐type enzyme, with the exception of the residue at position 141. Thus, the differences in 4‐methylpyrazole binding between the mutant and wild‐type σσ ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class‐specific differences in 4‐methylpyrazole binding to the human ADH isoenzymes.
Thermal stability of hydrophobic heme pocket variants of oxidized cytochrome c
Authors: Liggins John R. , Nall Barry T. , Lo Terence P. , Brayer Gary D.
Published: February 2009

Microcalorimetry has been used to measure the stabilities of mutational variants of yeast iso‐1 cytochrome c in which F82 and L85 have been replaced by other hydrophobic amino acids. Specifically, F82 has been replaced by Y and L85 by A. The double mutant F82Y, L85A iso‐1 has also been studied, and the mutational perturbations are compared to those for the two single mutants, F82Y iso‐1 and L85A iso‐1. Results are interpreted in terms of known crystallographic structures. The data show that (1) the destabilization of the mutant proteins is similar in magnitude to that which is theoretically predicted by the more obvious mutation‐induced structural effects; (2) the free energy of destabilization of the double mutant, F82Y, L85A iso‐1, is less than the sum of those of the two single mutants, almost certainly because, in the double mutant, the ‐OH group of Y82 is able to protrude into the cavity formed by the L85A substitution. The more favorable structural accommodation of the new ‐OH group in the double mutant leads to additional stability through (1) further decreases in the volumes of internal cavities and (2) formation of an extra protein‐protein hydrogen bond.
The mechanism of aconitase: 1.8 Å resolution crystal structure of the S642A:citrate complex
Authors: Lloyd S. J. , Lauble H. , Prasad G. S. , Stout C. D.
Published: February 2009

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 Å resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser → Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis‐aconitate, flip over by 180° about the Cα‐Cβ double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.