bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic probes for the α‐factor receptor
Authors: Naider Fred , Yaron Arieh , Ewenson Ariel , Tallon Michael , Xue Chu‐Biao , Srinivasan Jaya V. , Eriotou‐Bargiota Effimia , Becker Jeffrey M.
Published: December 2004

The binding of the tridecapeptide yeast mating phermone, α‐factor, to its receptor represents an excellent model for the investigation of peptide hormone–receptor interactions. In this paper we present a number of strategies to probe the binding site of the α‐factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the α‐ or ε‐amine in [Nle12]‐α‐factor was accomplished using 3‐[3,5‐diiodo‐4‐hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton‐Hunter reagent). At pH 8.0 in a N‐N‐dimethylformamide/water mixture the ratio of ε‐ to α‐acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2‐dimethyoxyethane/water mixture α‐acylation was favored by more than 3 to 1. The product distribution was sound to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially α‐acylated [Nle12]‐α‐factor and served as a temporary masking group during the synthesis of ε‐Bolton–Hunter acylated phermone. Biotin or diiodo Bolton–Hunter reagents were also directly incorporated into [Nle12]‐α‐factor or Lys[Nle12]‐α‐factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylace‐tamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton–Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays. These studies provide insights into the structural requirements for binding of α‐factor to its receptor and form a basis for detailed investigation of the receptor using biochemical procedures.
The vacuum UV CD bands of repeating DNA sequences are dependent on sequence and conformation
Authors: Gray Donald M. , Johnson Kenneth H. , Vaughan Marilyn R. , Morris Patricia A. , Sutherland John C. , Ratliff Robert L.
Published: December 2004

CD spectra were obtained for eight synthetic double‐stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A‐C) · d(G‐T)] and poly[d(A‐G) · d(C‐T)], (b) poly[d(A‐C‐C) · d(G‐G‐T)] and poly[d(A‐C‐G) · d(C‐G‐T)], and (c) poly[d(A) · d(T)], poly[d(A‐T) · d(A‐T)], poly[d(A‐A‐T) · d(A‐T‐T)], and poly[d(A‐A‐T‐T) · d(A‐A‐T‐T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G · C)‐containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180–191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right‐handed conformations. Among the set of sequences containing only A · T base pairs, poly[d(A) · d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A · T)‐rich polymers and was absent from two first‐neighbor estimations of the poly[d(A) · d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A · T)‐rich polymers was due in part of the fact that poly[d(A) · d(T)] exists in a noncanonical B conformation.
Synthesis, crystal structure, and molecular conformation of peptide N‐Boc‐L‐Pro‐dehydro‐Phe‐L‐Gly‐OH
Authors: Patel H. C. , Singh T. P. , Chauhan V. S. , Kaur P.
Published: December 2004

The peptide N‐Boc‐L‐Pro‐dehydro‐Phe‐L‐Gly‐OH was synthesized by the usual workup procedure and finally coupling the N‐Boc‐L‐Pro‐dehydro‐Phe to glycine. The peptide crystallizes in monoclinic space group P21 with a = 8.951(4) Å, b = 5.677 (6) Å, c = 21.192(11) Å, β = 96.97(4)°, V = 1069(1) Å3, Z = 2, dm = 1.295(5) Mgm−3, and dc = 1.297(4) Mgm−3. The structure was determined by direct methods using SHELXS86. The structure was refined by the block‐diagonal least‐squares procedure to an R value of 0.074 for 1002 observed reflections. The C–C distance of 1.33(2) Å is an appropriate double bond length. The angle C–C–C is 133(1)°. The peptide backbone torsion angles are θ1 = −167(1)°, ω0 = 179(1)°, ϕ1 = −48(1)°, ψ1 = 137(1)°, ω1 = 175(1)°, ϕ2 = 65(2)°, ψ2 = 15(2)°, ω2 = −179(1)°, and ϕ3 = −166(1)°. These values show that the Boc group has a trans–trans conformation while the peptide backbone adopts a β‐turn II conformation, which is stablized by an intramolecular hydrogen bond of length of 3.05(1) Å. The structures of dehydro‐Phe containing peptides suggest that the dehydro‐Phe promotes the β‐turn II conformation. The five‐membered pyrrolidine ring of the Pro residue adopts an ideal Cγ‐exo conformation with torsion angles χ = −24(1)°, χ = 34(1)°, χ = −30(1)°, χ = 15(1)°, and θ = 6(1)°. The side chain torsion angles in dehydro‐Phe are χ = −1(2)°, χ = −176(1)°, and χ = 8(2)°. The Plane of C–C–C is rotated with respect to the plane of the phenyl ring at 7(1)°, which indicates that the atoms of the side chain of dehydro‐Phe are essentially coplanar. The molecules form a 21 screw axis related hydrogen‐bonded rows along the b axis.
Kinetics of cooperative ligand–lattice binding: Fast Monte Carlo integration
Authors: Reiter Johannes , Epstein Irving R.
Published: December 2004

A fast Monte Carlo integration algorithm with varying time step is described for cooperative binding of ligands of arbitrary length to a one‐dimensional lattice. This algorithm is particularly suitable for strongly cooperative or anticooperative systems, i.e., when the time scales for different kinetic events are very different. As an application, the kinetics of a bimodal two‐ligand system are briefly discussed.
The binding of substituted cis‐Pt(II)‐diammines to duplex DNA
Authors: McCarthy Sharon L. , Hinde Robert J. , Miller Kenneth J. , Anderson Janet S. , Basch Harold , Krauss Morris
Published: December 2004

The results of a study of the binding to DNA of substituted cis‐Pt(II) diammines, (cis‐DP) are presented. Computer modeling of a series of cis‐Pt(NH2R)—where R = H, CH3, cyclopropyl, cyclobutyl, and cyclopentyl—to N7(G) atoms of two adjacent intrastrand guanine bases in a square planar complex in a pentamer duplex of DNA were performed. The stability of the complexes is studied by calculating the relative conformational energy of the cis‐DP–DNA complexes with molecular mechanics (MM) and the intrinsic binding energy, which is the relative binding energy for ligand replacement in the presence of the substituents R with quantum mechanics. In the model, the receptor site geometry and the conformation of the DNA is changed little in the accommodation of the series of monosubstituted diammines. These diammines bind to one family of DNA conformations, denoted as IC in a previous study, and this suggests that a common conformational feature in the DNA may exist to explain the smooth trend in activity. The slight increase in van der Waals energy resulting from an increasing number of atoms in the substituents is countered by a larger decrease in the ligand replacement energy as the substituent increases in size. This overall decrease in relative energy is consistent with the slight decrease in activity as the substituent size increases.
1H‐Nmr stereospecific assignments by conformational data‐base searches
Authors: Nilges Michael , Clore G. Marius , Gronenborn Angela M.
Published: December 2004

A search procedure is described for making stereospecific assignments at prochiral centers in proteins on the basis of nuclear Overhauser enhancement and coupling constant data derived from nmr experiments. A data base comprising torsion angles, associated 1H‐1H coupling constants and interproton distances is searched by a computer algorithm for sets of values that match the experimental data within specified error limits. Two different data bases are used. The first is a crystallographic data base derived from 34 well‐refined crystal structures; the second is a systematic data base derived from conformations of a short peptide fragment with idealized geometry by systematically varying the ϕ,ψ, and χ1 torsion angles. Both approaches are tested for β‐methylene groups with model data obtained from 20 crystal structures. The results for the two methods are similar though not identical, so that a combination of the two methods appears to be useful. With an appropriate choice of error estimates, around 80% of the β‐methylene groups could be assigned in the test calculations. In addition, results with experimental nmr data indicate that a similar percentage of stereospecific assignments can be made in practical situations.
A conformational comparision of two stereoisomeric cyclic dermorphin analogues employing nmr and computer simulations
Authors: Mierke Dale F. , Schiller Peter W. , Goodman Murray
Published: December 2004

In a continuation of our program to study the structure–activity relationship of peptide opiates, we report the conformational analysis of two cyclic tetrapeptides related to dermorphin—Tyr‐c[D‐Orn‐Phe‐Asp]‐NH2 and Tyr‐c[D‐Asp‐Phe‐Orn]‐NH2. These analogues have similar binding properties marked by a high selectivity for the μ‐opioid receptors because of a drastic decrease in the affinity for the δ‐opioid receptor. The conformational preferences of these analogues of dermorphin determined from proton nmr, molecular dynamics, and energy minimizations are quite similar. The constraint of the 13‐membered ring formed from cyclization is quite evident from the conformational analysis. The constrained ring system acts as a template maintaining the relative orientation of the exocyclic tyrosine and side chain of phenylalanine. Two intramolecular hydrogen bonds measured for the D‐Orn analogue in DMSO were disrupted upon the addition of water. For the D‐Asp analogue, two intramolecular hydrogen bonds were found stable in DMSO and water. The global conformations of the two peptides determined from nuclear Overhauser effects did not change with the solvent titration. The difference in the hydrogen bonding within the 13‐membered ring may account for the slight differences observed in the efficacy of the analogues at the μ‐opioid receptors.
Determination of solution conformation of DNA backbone: Application of homonuclear (J, δ) spectroscopy
Authors: Hosur R. V. , Chary K. V. R. , Saran Anil , Govil Girjesh , Miles H. Todd
Published: December 2004

Homonuclear two‐dimensional (J,δ) proton spectroscopy has been suggested as a method for the measurement of 1H‐31P coupling constants in oligonucleotides. The technique has been applied to a dinucleoside monophosphate G2′p5′C and a deoxydecanucleotide d(ACATCGATGT). PCILO energy calculations have been carried out to find minimum energy conformations with respect to the DNA backbone torsion angle ε, and these have been considered for the interpretation of the observed H3′‐31P coupling constants in oligonucleotides.
Disaccharide conformational flexibility. II. Molecular dynamics simulations of sucrose
Authors: Tran V. H. , Brady J. W.
Published: December 2004

Molecular dynamics simulations have been used to study the motions in vacuum of the disaccharide sucrose. Ensembles of trajectories were calculated for each of the five local minimum energy conformations identified in the adiabatic conformational energy mapping of this molecule. The model sucrose molecules were found to exhibit a variety of motions, although the global minimum energy conformation was found to be dynamically stable, and no transitions away from this structure were observed to occur spontaneously. In all but one of these vacuum trajectories, the intramolecular hydrogen bond between residues was maintained, in accord with recent nmr studies of this molecule in aqueous solution. Considerable flexibility of the furanoid ring was found in the trajectories. No “flips” to the opposite puckering for this ring were found in the simulations starting from the global minimum, although such a transition was observed for a trajectory initiated with one of the higher local minimum energy conformations. Overall, the observed structural fluctuations were consistent with the experimental picture of sucrose as a relatively rigid molecule.
The interaction of cyclosporin and calmodulin
Authors: Steiner Robert F. , Albaugh Sharon
Published: December 2004

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2+‐dependent and 1 : 1 Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys‐27 of wheat germ calmodulin suggest that the primary binding site is not located on the N‐terminal lobe (residues 1–65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N‐terminal half‐molecule (residues 1–77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.
X‐ray diffraction studies on the cross‐β form of poly (O‐benzyl‐L‐serine)
Authors: Takeda Yasuhiro
Published: December 2004

Poly(O‐benzyl‐L‐serine) of degree. polymerization 90 in oriented film cast from dichloroacetic acid (DCA) solution yielded the x‐ray patterns of the intramolecular cross‐β form, in which the extended segments stand upward and downward to the surfaces of a thin film. These x‐ray photographs further suggest that residual solvent molecules are bound to the polymer, and it may provide an important clue in understanding the nature of the cross‐β conformation in a diluted DCA solution.
Contribution of asymmetric ligand binding to the apparent permanent dipole moment of DNA
Authors: Plum G. Eric , Bloomfield Victor A.
Published: December 2004

Despite its antiparallel symmetry, DNA often appears to possess a permanent electric dipole moment in transient electro‐optical experiments. We propose that this may be due to the asymmetric binding of charged ligands to the DNA. We have used the fluctuating dipole theory of Kirkwood and Shumaker to calculate the contribution of asymmetric ligand binding to the electro‐optic orientation function, and Monte Carlo computer simulation to calculate the reversing pulse behavior, as a function of ligand binding density. The results indicate that the effect should be observable even against the background of the sizable induced dipole moment produced by polarization of the counterion atmosphere.
The effects of hydration on the dynamic mechanical properties of elastin
Authors: Lillie M. A. , Gosline J. M.
Published: December 2004

The dynamic mechanical properties of elastin have been quantified over a temperature and hydration range appropriate for a biological polymer. Composite curves of the tensile properties at constant water contents between 28.1 and 44.6% (g water/100 g protein) were typical of an amorphous polymer going through its glass transition. Water content had no effect on the shape of the curves, but shifted them a distance aC along the frequency axis. The combined effects of hydration and temperature are given in a series of isoshift curves where elastin's properties are constant along any one curve. A 1% change in hydration has the same effect as a 1°–2° change in temperature, depending on the initial water content and temperature. Theoretical isoshift curves that matched the experimental data were predicted using the WLF equation and coefficients determined from the data. These data form a basis to predict the role of elastin in arterial disease based on changes in its chemical and physical environment.
Theory for the extension of a linear polyelectrolyte attached at one end in an electric field
Authors: Schurr J. Michael , Smith Steven B.
Published: December 2004

A simple theory is developed for the extension of a uniformly charged linear polyion attached at one end in an electric field. For a polyion consisting of a very large number (N) of Kuhn lengths (b), the mean extension in the direction of the electric field (E) is given accurately by Rz = (b/A) In (sinh (NA)/NA), where A = EQb/kBT and Q is the effective charge associated with each Kuhn length. For any value of E, no matter how small, a polyion of sufficient length, such that NA ≫ 1.0, will be essentially fully extended. When A ≪ 1.0, as is the case for DNA in weak electric fields, the head end of the chain is only weakly oriented, even though the stem and tail may be completely aligned. In the linear regime, NA < 1.0, Rz is proportional to E, Q, and N2. The linear density of chain elements is calculated for a chain in the linear regime. This theory provides a basis to determine the product Qb from direct microscopic measurements of Rz vs. E for fluorescent‐labeled circular DNAs that are topologically snared near voids or surfaces of gels. Experimental realization of this suggestion is described in the following paper.
Langevin modes of macromolecules: Applications to crambin and DNA hexamers
Authors: Kottalam J. , Case D. A.
Published: December 2004

Langevin modes described the behavior of atoms moving on a harmonic potential surface subject to viscous damping described by a classical Langevin equation. We present applications to the protein crambin and to the DNA duplex d(CpGpCpGpCpG)2 and its complex with ethidium. Our friction matrix is weighted according to surface area exposed to solvent, and results are reported for various values of the solvent viscosity and models for hydro‐dynamic interactions. Even for relatively small solvent friction (η = 0.3 cp) a substantial number of modes are overdamped, and time correlation functions decay smoothly without the oscillations characteristic of gas‐phase calculations. Perturbation theory starting from the gas‐phase modes is accurate for many low‐frequency modes (which are overdamped in the presence of solvent), but fails badly for higher modes. For correlation functions of interest to fluorescence depolarization or nmr relaxation, the plateau values are insensitive to solvent viscosity, but the relaxation times are not. The advantages and limitations of this analysis of macromolecular motions are discussed.
Cooperativity of carbohydrate moiety orientation and β‐ turn stability is determined by intramolecular hydrogen bonds in protected glycopeptide models
Authors: Hollosi M. , Perczel A. , Fasman G. D.
Published: December 2004

The 2, 3, 4, 6‐Tetra‐O‐acetyl‐β‐D‐gluco‐, and β‐D‐galactopyranosides, as well as approximately 4 : 1 anomeric mixtures of α‐ and β‐mannopyranosides of Boc‐X‐Y‐NHCH3 dipeptides (X‐Y = Pro‐Ser, Pro‐D‐Ser, Val‐Ser, Val‐D‐Ser, and Gly‐Ser) have been synthesized. CD and ir spectroscopic studies were performed to characterize the conformation of the glycosylated peptide backbone and examine the possible formation of intrapeptide and glycopeptide intramolecular H‐bonds. It was found that O‐glycosylated peptides containing a D‐serine residue are likely to adopt a type II β‐turn while those with the Pro‐Ser or Val‐Ser sequence feature a type I(III) β‐turn in solution. Glycosylation also increases the magnitude of the CD bands, characteristic of the given type of β‐turns, which can be interpreted as an indication of the stablization of the folded backbone conformation. Infrared data showed that in nonpolar solutions the peracetyl glycopeptides adopt both single‐ and double H‐bonded conformations whose ratio, in some cases, depends on the position at C‐2′ of the H‐bond acceptor acetoxy group. These data suggest that five‐, seven‐, or ten‐membered glyco‐turns may play an important role in fixing the steric orientation of the carbohydrate antennae systems in glycoproteins.
A molecular dynamics investigation of the elastomeric restoring force in elastin
Authors: Wasserman Z. R. , Salemme F. R.
Published: December 2004

A repetitive polypentapeptide organized as a connected chain of β‐bends is believed to be an important structural element of elastin, the major elastomer in biological systems. Molecular dynamics simulations were carried out on hydrated polymers of (Val‐Pro‐Gly‐Val‐Gly)18 at various extensions. Analysis of the fluctuations of backbone angles in relaxed elastin showed that particularly large‐amplitude torsional motions occur in ϕ and ψ angles of residues connecting sequentially adjacent hairpin bends. Many such motions reflect peptide plane librations that result from anticorrelated crankshaft rotations of ψi and ϕi+1. These effects were much reduced in stretched polymer models. The conformational entropy of relaxed and stretched elastin models was estimated using a treatment due to Meirovitch, and gave a calculated decrease in entropy of about 1 cal/mol deg when the polymer was stretched to 1.75 times its original length. There are large changes in solvent‐accessible surface area during the intitial stages of elastin stretching. Collectively these results suggest that hydrophobic interactions make contributions to elastin entropy at low extensions, but that librational mechanisms make large contributions to the elastic restoring force at longer extensions.
Determining minimum energy conformations of polypeptides by dynamic programming
Authors: Vajda Sandor , Delisi Charles
Published: December 2004

A combinatorial optimization approach is used for solving the multiple‐minima problem when determining the low‐energy conformations of short polypeptides. Each residue is represented by a finite number of discrete states corresponding to single residue local minima of the energy function. These precomputed values constitute a search table and define the conformational space for discrete minimization by a generalized dynamic programming algorithm that significantly limits the number of intermediate conformations to be generated during the search. Since dynamic programming involves stagewise decisions, it results in buildup‐type procedures implemented in two different forms. The first procedure predicts a number of conformations by a completely discrete search and these are subsequently refined by local minimization. The second involves limited continuous local minimization within the combinatorial algorithm, generally restricted to two dihedral angles in a buildup step. Both procedures are tested on 17 short peptides previously studied by other global minimization methods but involving the same potential energy function. The discrete method is extremely fast, but proves to be successful only in 14 of the 17 test problems. The version with limited local minimization finds, however, conformations in all the 17 examples that are close to the ones previously presented in the literature or have lower energies. In addition, resutls are almost independent of the cutoff energy, the most important parameter governing the search. Although the limited local minimization increases the number of energy evaluations, the method still offers substantial advantages in speed.
Structural versatility of peptides from Cα,α‐disubstituted glycines: Preferred conformation of the Cα,α‐diphenylglycine residue
Authors: Crisma M. , Valle G. , Bonora G. M. , De Menego E. , Toniolo C. , Lelj F. , Barone V. , Fraternal F.
Published: December 2004

The preferred conformation of the Cα,α‐diphenylglycine residue was determined in simple derivatives and dipeptides. The dipeptides were synthesized by the 5(4H)‐oxazolone (from the N‐para‐bromobenzoylated amino acid) method. This activated intermediate and a reaction by‐product, para‐bromobenzoylbenzhydrylamine, were characterized inter alia by x‐ray diffraction. Conformational energy calculations on the Cα,α‐diphenylglycine mono‐peptide, Ac‐Døg‐NHMe, indicate that this Cα,α‐symmetrically disubstituted residue is conformationally restricted and that its minimum energy conformation falls in the fully extended (C5) region. The results of the theoretical analysis are in agreement with the solution and crystal‐state structural tendency of mClAc‐Døg‐OH, Z‐Døg‐OtBu, pBrBz‐Døg‐Gly‐OMe and its tert‐butyl ester analogue, determined by ir absorption, lH‐nmr, and x‐ray diffraction, and also described in this work. The implications for the use of the Døg residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.
Mechanical properties of DNA films
Authors: Gevorkian S. G. , Khudaverdian E. E.
Published: December 2004

The Young's dynamical modulus (E) and the DNA film logarithmic decrement (v) at frequencies from 50 Hz to 20 kHz are measured. These values are investigated as functions of the degree of hydration and temperature. Isotherms of DNA film hydration at 25°C are measured. The process of film hydration changing with temperature is studied. It is shown that the Young's modulus for wet DNA films (E = 0.02–0.025 GN m−2) strongly increases with decreasing hydration and makes E = 0.5–0.7 GN m−2. Dependence of E on hydration is of a complex character. Young's modulus of denatured DNA films is larger than that of native ones. All peculiarities of changing of E and v of native DNA films (observed at variation of hydration) vanish in the case of denatured ones. The native and denatured DNA films isotherms are different and depend on the technique of denaturation. The Young's modulus of DNA films containing > 1 g H2O/g dry DNA is found to decrease with increasing temperature, undergoing a number of step‐like changes accompanied by changes in the film hydration. At low water content (< 0.3 g H2O/g dry DNA), changing of E with increasing temperature takes place smoothly. The denaturation temperature is a function of the water content.
A hydrodynamic study with quasielastic light scattering and sedimentation of bacterial elongation factor EF‐Tu · guanosine‐5′‐diphosphate complex under nonassociating conditions
Authors: Sam Tom , Mandel Michel , Pley Cornelis
Published: December 2004

The hydrodynamics of the bacterial elongation factor EF‐Tu have been studied in the presence of its ligand guanosine‐5′‐diphosphate (GDP) by sedimentation in the ultracentrifuge and quasielastic light scattering. Sedimentation studies have made it possible to establish experimental conditions under which only negligible aggregation of the protein occurs (neutral pH, concentration < 3 mg/mL). Analysis of the light intensity autocorrelation functions under these conditions revealed two independent scattering species with diffusion coefficients of 0.71 × 10−6 and 0.04 × 10−6 cm2 s−1. The material with the lower diffusion coefficient, i.e., the aggregates, represented less than 1% of the total number of EF‐Tu particles. The other 99% diffused as monomeric molecules with a molar mass corresponding to the value calculated from the known primary structure of the protein. The hydrodynamic parameters derived from the experimental data suggest that EF‐Tu·GDP in solution is close to a spherical particle.
Global fluctuations of the immunoglobulin domains under physiological conditions
Authors: Kawata Yasushi , Hamaguchi Kozo
Published: December 2004

Hydrogen‐exchange rates of the indole NH proton of a tryptophan residue, buried fully in the interior of each of the constant (CL) and variable (VL) fragments of a type‐κ‐immunoglobulin light chain, were studied at various pH values and at 25°C under 1H‐nuclear magnetic resonance. The activation energies for the exchange reactions were determined also and compared with those for the unfolding reactions of these fragments induced by guanidine hydrochloride. The pH profiles of the exchange rates of the CL(κ) and VL(κ) fragments were very similar to that for a CL(λ) fragment reported previously.4 It was found that the CL(κ) and VL(κ) fragments as well as the CL(λ) fragment undergo a global unfolding transition with a conformation very similar to that of the fully unfolded state induced by guanidine hydrochloride even under physiological conditions.
Structure of the hydration shells of oligo(dA‐dT)·oligo(dA‐dT) and oligo(dA)·oligo(dT) tracts in B‐type conformation on the basis of Monte Carlo calculations
Authors: Eisenhaber F. , Tumanyan V. G. , Abagyan R. A.
Published: December 2004

Monte Carlo simulations [(N, V, T)‐ensemble] were performed for the hydration shell of poly(dA‐dT)·poly(dA‐dT) in canonical B form and for the hydration shell of poly(dA) ·poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero‐inclined base pair plane (B′ conformation). We introduced helical periodic boundary conditions with a rather small unit cell and a limited number of water molecules to reduce the dimensionality of the configuration space. The coordinates of local maxima of water density and the properties of one‐ and two‐membered water bridges between polar groups of the DNA were obtained.

The AT‐alternating duplex hydration mirrors the dyad symmetry of polar group distribution. At the dApdT step, a water bridge between the two carbonyl oxygens O2 of thymines is formed as in the central base‐pair step of Dickerson's dodecamer. In the major groove, 5‐membered water chains along the tetranucleotide pattern d (TATA) · d (TATA) are observed.

The hydration geometry of poly (dA) · poly(dT) in canonical B conformation is distinguished by autonomous primary hydration of the base‐pair edges in both grooves. When this polymer adopts a conformation with highly inclined bases and narrow minor groove, the water density distribution in the minor groove is in excellent agreement with Dickerson's spine model. One local maximum per base pair of the first layer is located near the dyad axis between adjacent base pairs, and one local maximum per base pair in the second shell lies near the dyad axis of the base pair itself. The water bridge between the two strands formed within the first layer was observed with high probability. But the water molecules of the second layer do not have a statistically favored orientation necessary for bridging first layer waters. In the major groove, the hydration geometry of the (A · T) base‐pair edge resembles the main features of the AT‐pair hydration derived from other sequences for the canonical B form. The preference of the B′ conformation for oligo(dA) · oligo(dT) tracts may express the tendency to common hydration of base‐pair edges of successive base pairs in the grooves of B‐type DNA.

The mean potential energy of hydration of canonical B‐DNA was estimated to be −60 to −80 kJ/mole nucleotides in dependence on the (G · C) contents. Because of the small system size, this estimation is preliminary.
Synthetic peptide helices in crystals: Structure and antiparallel and skewed packing motifs for α‐helices in two isomeric decapeptides
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Uma K. , Balaram P.
Published: December 2004

The isomeric decapeptides Boc‐Aib‐Ala‐Leu‐Ala‐Aib‐Aib‐Leu‐Ala‐Leu‐Aib‐OMe (II) and Boc‐Aib‐Ala‐Aib‐Ala‐Leu‐Ala‐Leu‐Aib‐Leu‐Aib‐OMe (III), are predominantly a‐helical with little effect on the conformation with interchange of Aib/Ala residues or Aib/Leu residues. The packing motif of helices in crystal II is antiparallel, whereas the helices pack in a skewed fashion in crystal III, with a 40° angle between neighboring helix axes. Crystal III contains a water molecule in a hydrophobic hole that forms hydrogen bonds with two carbonyl oxygens that also participate in 5 → 1 type hydrogen bonds. Values for helical torsional angles ϕ and ψ assume a much wider range than anticipated. Crystal II: C49H88N10O13, space group P21 with a = 16.625(2) Å, b = 9.811(5) Å, c = 18.412(3) Å, β = 99.79(1)°, Z = 2, R = 5.7% for 4338 data with |F0 | > 3σ(F). Crystal III: C49H88N10O13. 1/2H2O, space group P21, with a = 11.072(2) Å, b = 34.663(5) Å, c = 16.446(3) Å, β = 107.85(1) °, Z = 4, R = 8.3% for 6087 data with |F0| > 3σ(F).
Randomness in the heparin polymer: Computer simulations of alternative action patterns of heparin lyase
Authors: Cohen David Marshall , Linhardt Robert J.
Published: December 2004

Heparin is a mixture of linear polysaccharides of undetermined sequence. Both biosynthetic data and computer simulation studies have established that each heparin polymer chain is comprised of oligosaccharides of defined sequence, representing ordered domains. One such ordered domain is a pentasaccharide corresponding to heparin's antithrombin III binding site. Previous computer simulation studies, performed under the assumption that heparin lyase (heparinase, EC 4.2.2.7), has a random endolytic action pattern, suggested that certain of these ordered oligosaccharide domains may themselves be nonrandomly arranged in the heparin polymer. The present, work presents computer simulations of alternative action patterns for heparin lyase while assuming a random distribution of these oligosaccharide units within the heparin polymer. We consider action patterns that are determined solely by the primary structure of the substrate molecules. Results of the simulations are compared to (1) the experimental measurements of product chains formed throughout the reaction and (2) the change in weight average molecular weight Mw as a function of reaction completion as determined by absorbance at 232 nm.

From the simulation of 60 action patterns for heparin lyase, we infer that one of the following statements concerning heparin and heparin lyase is true: (1) Heparin is a random arrangement of a small number of structurally defined oligosaccharide units. Heparin lyase changes its action pattern during the depolymerization of heparin (perhaps influenced by the secondary structure of substrate). (2) Heparin contains clusters of oligosaccharide sequences that are present in low concentrations (overall) in the polymer. Heparin lyase has a specificity for cleaving glycosidic linkages either exolytically at the nonreducing terminus of a chain or (endolytically) at the reducing side of these rare oligosaccharide sequences.
Environment‐dependent conformation of Boc‐Pro‐Ser‐NHCH3
Authors: Perczel A. , Hollósi M. , Fülöup V. , Kálmán A. , Sándor P. , Fasman G. D.
Published: December 2004

The conformation of Boc‐Pro‐Ser‐NHCH3 (1) was studied in the solid state and in solution. In the crystal, the steric structure of 1 is characterized by an E(cis) urethane tertiary amide bond and the lack of intramolecular H bonds. Four‐hundred megahertz 1H‐ and 101‐MHz 13C‐nmr studies in CDCl3 clearly show the presence of two conformers differing in the rotameric state of the tertiary‐amide bond. Selective 1H‐13C nuclear Overhauser enhancement experiments at −20°C as well as 1H‐nmr and ir data indicate that the major trans conformer (84% in CDC13) may adopt a type I β‐turn conformation with a possible Oγ  H‐N interaction, similarly to Piv‐Pro‐Ser HCH3 (2) [A. Aubry, N. Ghermani, and M. Mar‐raud (1984) Int. J. Peptide Protein Res. 23, 113‐122]. Molecular mechanics calculations on the cis rotamer show that the β‐pleated‐like backbone conformation of serine in the crystal of 1 is not a low‐energy state for the isolated molecule; it is caused by packing forces, particularly by the helical network of intermolecular H bonds.
Poly[d(Gm5C)·d(Gm5C)] can assume the Z′ form: A CD study
Authors: Zhong Lingxiu , Johnson W. Curtis
Published: December 2004

Poly [d(Gm5C)·d(Gm5C)] can be titrated from the Z form in 30% ethanol to the Z′ form by adding either ethanol or divalents. Analysis by singular‐value decomposition of CD spectra recorded during the titration reveal that ethanol and transition metal induced changes are two state with a single‐step titration curve. When the change is induced by arkaline earth metals it is still two state, but in contrast the titration curve is complex, indicating two binding sites.
Correlation between dinucleotide periodicities and nucleosome positioning on mouse satellite DNA
Authors: Lowman Henry , Bina Minou
Published: December 2004

Previous studies demonstrated 16 well‐defined nucleosome locations (A–P) on a tandemly repeated prototype 234 base pair (bp) mouse satellite repeat unit. We have aligned the A–P fragments to search for DNA sequence elements that might contribute to nucleosome placement at these positions. Our results demonstrate a strikingly regular, uninterrupted, periodic pattern for the AA dinucleotide occurrences along the entire length of the aligned fragments. The periodicity of the AA occurrences is about 9.7 bp. The pattern exhibits a local minimum at position 74, near the nucleosome dyad axis of symmetry. Other dinucleotides—including AC : GT, CA : TG, and CC : GG—are also placed periodically, but their patterns of occurrence are less regular and less frequent than AA. The calculated spacings between consecutive preferred nucleosome locations on mouse satellite DNA are nearly identical, corresponding to multiples of 9.7 bp. The correlation between the periodicity of dinucleotide occurrences and the average spacing of nucleosome positions suggests that the preferred nucleosome locations recur at intervals that may correspond to the DNA helical repeat in the mouse satellite nucleosomes, and that the histone octamers sample (or slip along) the duplex in steps of 9.7 bp during nucleosome formation on mouse satellite DNA.
The thermal denaturation of nonpolymerizable αα‐tropomyosin and its segments as a function of ionic strength
Authors: Mo Jianming , Holtzer Marilyn Emerson , Holtzer Alfred
Published: December 2004

Nonpolymerizable tropomyosin (NPTm) is found to unfold thermally at high ionic strength almost exactly as the parent protein, but it does not aggregate at low ionic strength. Thus, NPTm can be used as a tropomyosin surrogate whose coiled‐coil structural stability can be probed by varying the ionic strength. Studies of NPTm by CD show that increasing ionic strength stabilizes the coiled‐coil structure. CD spectra over a wide range of helix content, obtained by varying either temperature or ionic strength, show an isodichroic point at 203 nm, suggesting a local, residue‐level, two‐state model. At given temperature, such a local helix ⇆ random equilibrium suggests In [Φh/(l − Φh)] = A1 + A2In, wherein Φh is the fraction helix, and A1, A2 and n are constants. In the low ionic strength region, theoretical limiting laws for ionic strength mediated charge–charge, dipole–dipole, and apolar–apolar (salting out) interactions give, respectively, n = 0.5, 1.0, and 1.0. Our experimental values for 40°C, where the data span a wide range of helix content, show n = 1.0, suggesting that ionic strength stabilizes either by reducing dipole–dipole repulsions or by enhancing hydrophobic interactions, both probably interhelix in nature. Two segments of tropomyosin, 11Tm127 and 142Tm281, neither of which aggregate at low ionic strength, give results similar to those for NPTm, i.e., n = 0.96 and 0.84, respectively.
The effect of the L‐azetidine‐2‐carboxylic acid residue on protein conformation. III. Collagen‐like poly(tripeptide)s
Authors: Zagari Adriana , Némethy George , Scheraga Harold A.
Published: December 2004

The chemical and biological properties of collagen are altered by the biosynthetic substitution of the L‐azetidine‐2‐carboxylic acid (Aze) residue in the place of proline. The reasons for this alteration have been studied by means of conformational energy computations on single‐ and triple‐stranded structures formed by poly (GIy‐X‐Y) poly(tripeptide)s, where X and Y can be Pro or Aze. The most stable triple helix formed by Poly (Gly‐Pro‐Aze) is collagen‐like, but all low‐energy triple helices that can be formed by poly (Gly‐Aze‐Pro) and poly (Gly‐Aze‐Aze) are very different from collagen. Thus, the regular substitution of Aze for Pro in position X is not compatible with the collagen structure. In the absence of solvent effects, all of these triple helices are stable, relative to the statistical coil, but the substitutions reduce the stability of the collagen‐like triple helix, as compared with poly (Gly‐Pro‐Pro).
α‐Helix to random coil transitions of two‐chain coiled coils: Experiments on the thermal denaturation of isolated segments of αα‐tropomyosin
Authors: Holtzer Marilyn Emerson , Holtzer Alfred
Published: December 2004

Circular dichroism (CD) experiments in the backbone (200–240 nm) region are reported for four isolated, excised two‐chain, coiled‐coil segments whose chains comprise, respectively, residues 11–127, 142–281, 1–189, and 190–284 of the rabbit αα‐tropomyosin (Tm) sequence. The uv and CD spectra for the noncross‐linked segments are very similar to those for parent Tm. At 3°C, all have a helix content of 90% or more; moreover, all thermal denaturation curves depend on concentration, as required by mass action, and are completely reversible. At comparable concentrations, solutions show values of T1/2 (the temperature at which the helix content is 50%) following the order of 11Tm127 ∼ 1Tm189 > 142Tm281 > 190Tm284. The thermal unfolding data for 11Tm127, 190Tm284, and 142Tm281 fall on apparently monophasic curves (single inflection point). However, curves for 1Tm189 show a heretofore unknown low temperature transition in which the helix content drops from ∼ 90% at 2°C to ∼ 73% at 20°C, indicating that this segment has one or more weak sections totaling approximately 50 residues per chain. Since thermal denaturation curves for noncross‐Iinked 11Tm127, 142Tm281, and Tm have no such low temperature transition, i.e., the helix content is not additive, the weak region probably comprises the bulk of the residues between 127 and 189 in 1Tm189, but is somehow stabilized in 142Tm281 and in parent Tm. This nonad‐ditivity may be explainable by a combination of lopp entropy and local adjustments in packing of hydrophobes in the helix–helix interface. For Cl90‐cross‐linked 142Tm281, the uv and CD spectra and helix content are very similar to those for other coiled‐coil species. In addition, the thermal unfolding curve shows a “pretransition” that is similar to but broader than that for cross‐linked parent Tm and a “posttransition” that is almost identical to that for cross‐linked parent Tm.
Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range
Authors: Makhatadze G. I. , Medvedkin V. N. , Privalov P. L.
Published: December 2004

The partial molar volumes of various compounds that model protein constituent groups, such as tripeptides (Gly‐X‐Gly, where X = Gly, Ala, Val, Leu, Ile, Pro, Met, His, Ser), homopeptides (Glyn, n = 3, 4, 5), and simple organic analogues of amino acid side chains (methanol, acetamide, propanamide, acetic acid, propanoic acid, n‐butanamine, n‐butanamine nitrate, n‐propylguanidine nitrate, 4‐methylphenol), have been determined in aqueous solution with a vibrational densimeter in the temperature range of 5–85°C. The partial molar volumes of amino acid side chains and the peptide unit were estimated from the data obtained. Assuming additivity of component groups, the partial molar volumes of polypeptide chains of several proteins over a broad temperature range were calculated. The partial molar volume functions of four proteins (myoglobin, cytochrome C, ribonuclease A, lysozyme) were compared with those determined experimentally for the unfolded and native forms of these proteins. It has been shown that the average deviation of the calculated functions from the experimental ones does not exceed 3% over the temperature range studied.
The influence of macromolecular crowding on thermodynamic activity: Solubility and dimerization constants for spherical and dumbbell‐shaped molecules in a hard‐sphere mixture
Authors: Berg Otto G.
Published: December 2004

Macromolecules in solution can have large effects on the properties of other solutes through nonideal excluded‐volume (crowding) interactions. Minton [(1983) Mol. Cell. Biochem. 55, 119–140] has calculated such effects by treating the macromolecules as a hard‐sphere fluid in a background of an inert structureless solvent. In the present paper these calculations are extended by including the primary solvent as a separate component in a hard‐sphere mixture. The results are in good agreement with experimental data. However, some predictions of this model differ drastically from those based on Minton's approach. Thus, much smaller effects from macromolecular crowding, particularly by smaller molecules, are expected. The present results also predict a much larger dependence on the shape of the molecules under study; notably for a dimerization reaction, it is found that the excluded‐volume effects actually can destabilize side‐by‐side binding of two spherical molecules, while a dimerization to a spherical complex is stabilized. Therefore there will exist intermediate shapes of complexes whose stability is insensitive to crowded‐volume effects. The consequences for crowding effects inside the living cell are also discussed.
Synthesis and peptide bond orientation in tetrapeptides containing L‐azetidine‐2‐carboxylic acid and L‐proline
Authors: Tsai F.‐H. , Overberger C. G. , Zand R.
Published: December 2004

The role of the amino acid proline in influencing the secondary and tertiary structure of proteins and polypeptides has been an area of active study for many years. We have investigated this problem by incorporating the four‐membered ring amino acid, azetidine‐2‐carboxylic acid, into some proline polypeptides. An adjunct to the synthesis of the peptides was the synthesis of azetidine‐2‐carboxylic acid and its resolution. We developed an improved synthesis of N‐benzhydryl‐2‐carbobenzyloxy azetidine, an essential intermediate required for the synthesis of Lazetidine‐2‐carboxylic acid. This amino acid was subsequently obtained via the partial hydrogenation of the N‐benzhydryl compound, under mild conditions. Our ability to isolate the intermediate N‐benzhydryl‐2‐carboxylic acid demonstrated that the rate of cleavage of the O‐benzyl ester group in this molecule is faster than the cleavage of the N‐benzhydryl group.

The tetrapeptides, Boc‐(LPro)3‐LAze‐Opcp, and Boc‐(LAze‐LPro)2‐Opcp (Boc: t‐butoxycarbonyl; Pro: praline; Aze: azetidine‐2‐carboxyl acid; Opcp: pentachlorophenyl), were prepared using traditional solution peptide synthesis. They were characterized by direct chemical ionization‐mass spectrometry, CD spectra, and 13C‐ and 1H‐nmr spectroscopy. The assessment of the secondary structure assessment of the two peptides using the methods noted above has led us to conclude that the compound Boc‐(L‐Aze‐LPro)2‐Opcp, in trifluoroethanol, has an all‐cis peptide bond conformation with ϕ and ψ torsion angles compatible with a left‐handed helix. The secondary structure assessment of the peptide Boc‐(LPro)3‐LAze‐Opcp, in chloroform or trifluoroethanol, leads to an assignment of both cis and trans peptide bonds as being present in the peptide. We have interpreted this latter finding as indicating that the introduction of the azetidine group into a peptide containing three consecutive praline residues in a linear sequence perturbs the normal proline peptide secondary structure in this tetrapeptide.