bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: Application to the DNA binding domain of lac repressor from Escherichia coli
Authors: de Vlieg J. , Scheek R. M. , van Gunsteren W. F. , Berendsen H. J. C. , Kaptein R. , Thomason J.
Published: May 2005

The technique of two‐dimensional nuclear magnetic resonance (2D‐NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom‐atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 “hydrogen bond” distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints.

Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.
Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30
Authors: Leijonmarck Marie , Appelt Krzysztof , Badger John , Liljas Anders , Wilson Keith S. , White Stephen W.
Published: May 2005

The structure of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and 1.30 from Bacilus Stearothermophilus display a remarkably similar fold in which alpaha‐helices pack onto one side of an antiparallel, three‐stranded, beta‐pleated sheet. A detailed comparison of the structures by least‐squares methods reveals that more than two‐thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 Å. The principal difference is an extra alpha‐helix in L12 CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.
Photochemical crosslinking of bacteriophage T4 single‐stranded DNA‐binding protein (gp32) to oligo‐p(dT)8: Identification of phenylalanine‐183 as the site of crosslinking
Authors: Shamoo Yousif , Williams Kenneth R. , Konigsberg William H.
Published: May 2005

Using ultraviolet light, both the 33,000‐dalton single‐standard DNA‐binding protein from T4 bacteriophage (gp32)as well as a 25,000‐dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single‐stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion‐exchange high‐performance liquid chromatography. Gas‐phase sequencing of this modified peptide gave the following sequence: Gln‐Val‐Ser‐Gly‐(X)‐Ser‐Asn‐Tyr‐Asp‐Glu‐Ser‐Lys, which corresponds to residues 179–190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:P(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.
Calculation of the total electrostatic energy of a macromolecular system: Solvation energies, binding energies, and conformational analysis
Authors: Gilson Michael K. , Honig Barry
Published: May 2005

In this report we describe an accurate numerical method for calculating the total electrostatic energy of molecules of arbitrary shape and charge distribution, accounting for both Coulombic and solvent polarization terms. In addition to the solvation energies of individual molecules, the method can be used to calculate the electrostatic energy associated with conformational changes in proteins as well as changes in solvation energy that accompany the binding of charged substrates. The validity of the method is examined by calculating the hydration energies of acetate, methyl ammonium, ammonium, and methanol. The method is then used to study the relationship between the depth of a charge within a protein and its interaction with the solvent. Calculations of the relative electrostatic energies of crystal and misfolded conformations of Themiste dyscritum hemerythrin and the VL domain of an antibody are also presented. The results indicate that electrostatic charge‐solvent interactions strongly favor the crystal structures. More generally, it is found that charge‐solvent interactions, which are frequently neglected in protein structure analysis, can make large contributions to the total energy of a macromolecular system.
Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase‐trimethoprim, a drug‐receptor system
Authors: Dauber‐Osguthorpe Pnina , Roberts Victoria A. , Osguthorpe David J. , Wolff Jon , Genest Moniqe , Hagler Arnold T.
Published: May 2005

A study of the binding of the antibacterial agent trimethoprim to Escherichia coli dihydrofolate reductase was carried out using energy minimization techniques with both a full, all‐atom valence force field and a united atom force field. Convergence criteria ensured that no significant structural or energetic changes would occur with further minimization. Root‐mean‐square (RMS) deviations of both minimized structures with the experimental structure with the experimental structure were calculated for selected regions of the protein. In the active site, the all‐atom minimized structure fit the experimental structure much better than did the united atom structure. To ascertain what constitutes a good fit, the RMS deviations between crystal structures of the same enzyme either from different species or in different crystal environments were compared. The differences between the active site of all‐atom minimized structure and the experimental structure are similar to differences observed between crystal structures of the same protein.

Finally, the energetics of ligand binding were analyzed for the all‐atom minimized coordinates. Strain energy induced in the ligand, the corresponding entropy loss due to shifts in harmonic frequencies, and the role of specific residues in ligand binding were examined. Water molecules, even those not in direct contact with the ligand, were found to have significant interaction energies with the ligand. Thus, the inclusion of at least one shell of waters may be vital for accurate simulations of enzyme complexes.
Oligopeptide biases in protein sequences and their use in predicting protein coding regions in nucleotide sequences
Authors: McCaldon Peter , Argos Patrick
Published: May 2005

We have examined oligopeptides with lengths ranging from 2 to 11 residues in protein sequences that show no obvious evolutionary relationship. All sequences in the Protein Identification Resource database were carefully classified by sensitive homology searches into superfamilies to obtain unbiased oligopeptide counts. The results, contrary to previous studies, show clear prejudices in protein sequences. The oligopeptide preferences were used to help decide the significance of sequence homologies and to improve the more general methods for detecting protein coding regions within nucleotide sequences.
Surface interactions of γ‐crystallins in the crystal medium in relation to their association in the eye lens
Authors: Sergeev Y. V. , Chirgadze Y. N. , Mylvaganam S. E. , Driessen H. , Slingsby C. , Blundell T. L.
Published: May 2005

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, γ‐II and γ‐IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of γ‐II as compared with 13% in γ‐III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in γ‐II, which are replaced by Met 103 and His 155 in γ‐IIIb. A similar substitution of these residues is observed in different gene products of γ‐crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of γ‐crystallins in the native medium of the lens.
Interaction of peptide boronic acids with elastase: Circular dichroism studies
Authors: Berry S. C. , Fink Anthony L. , Shenvi A. B. , Kettner C. A.
Published: May 2005

Boronic acid derivatives of good peptide substrate of the serine proteases cause slow‐binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol. Chem. 259:15106–15114, 1984). These inhibitors are thought to act as reaction‐intermediate analogs. Three peptides Boronic acids—Ac‐Pro‐boro‐Val‐OH, DNS‐Ala‐Pro‐boro‐Val‐OH, and Ac‐Ala‐Ala‐Pro‐boro‐Val‐OH—were chosen for farultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitors (Ki = 0.27 μM) and the latter are slow‐binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel β‐sheet by the peptide inhibitors itself indicate that there is no significant change in the secondary structure of the enzyme in the either the initial or final inhibitors complex. A kinetic experiment confirmed that the slow‐binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the sow binding.
Diffusion‐collision model for the folding kinetics of myoglobin
Authors: Bashford Donald , Cohen F. E. , Karplus M. , Kuntz I. D. , Weaver D. L.
Published: May 2005

The diffusion‐collision model has been used to analyze the folding kinetics of myoglobins. The microdomains, which are the basic units that coalesce during the folding, are identified with the helices and the stabilizing contacts between helices are determined form the native structure. Both association and disassociation reactions are included and a range of stabilization parameters is investigated to determine the variations in overall rate and the relative contributions made by the different intermediates during the folding process. In a comparison of folding to the native state and to the midpoint of the folding transitions. (i.e., 50% native protein at the completion of the reaction) significant differences in the contributing intermediates are found.
Further experimental studies of the disulfide folding transition of ribonuclease A
Authors: Wearne Steven J. , Creighton Thomas E.
Published: May 2005

Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate‐limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three‐disulfide intermediates. (2) A multiple pathway mechanism with different rate‐limiting steps for each pathway. The various rate‐limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding.

Refolding of reduced ribonuclease A requires three‐disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one‐ and two‐disulfide intermediate species are populated. Therefore, no rate‐limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all‐or‐none process; no disulfide intermediates accumulate to detectable to detectable levels or precede the rate‐limiting step. Mechanism (2) requires that such intermediates precede the rate‐limiting step and accumulate to substantial levels.

The different proposal were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation are breakage.
Further studies of the helix dipole model: Effects of a free α‐NH3+ or α‐COO− group on helix stability
Authors: Fairman Robert , Shoemaker Kevin R. , York Eunice J. , Stewart Jhon M. , Baldwin Robert L.
Published: May 2005

Interactions between the α‐helix peptide dipoles and charged groups close to the ends of the helix were found to be an important determinant of α‐helix stability in a previous study.1 The charge on the N‐terminal residue of the C‐peptide from ribonuclease A was varied chiefly by changing the α‐NH2 blocking group, and the correlation of helix stability with N‐terminal charge was demonstrated. An alternative explanation for some of those results is that the succinyl and acetyl blocking groups stabilize the helix by hydrogen bonding to an unsatisfied main‐chain NH group. The helix dipole model is tested here with peptides that contain either a free α‐NH α‐COO− groups, and no other charged groups that would titrate with similar pKa's. This model predicts that α‐NH3α‐COO‐ groups are helix‐destabilizingand that the destabilizing interactions are electrostatic in origin. The hydrogen bonding model predicts that α‐NH3 and α‐COO‐ groups are not themselves helix‐destabilizing, but that an acetyl or amide blocking group at the N‐ or C‐ terminus, respectively, stabilizes the helix by hydrogen bonding to an unsatisfied main‐chain NH or CO group.

The results are as follows: (1) Removal of the charge from α‐NH3 and α‐COO‐ groups by pH titration stabilizes an α‐helix. (2) The increase in helix stability on pH titration of these groups is close to the increase produced by adding an acetyl or amide blocking group. (3) The helix‐stabilizing effect of removing the charge from α‐NH3 and α‐COO‐ groups by pH titration is screened by increasing the NaCl concentration, and therefore the effect is electrostatic in origin. (4) Replacing the C‐terminal amide blocking group with a methylester blocking group, which cannot donate a hydrogen bond, causes little change in helix stability.
Effect of protein conformation on rate of deamidation: Ribonuclease A
Authors: Wearne Steven J. , Creighton Thomas E.
Published: May 2005

The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determine. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds.

Deamidation of the labile Asn‐67 residue of RNase A was followedelectrophoretically and chromatographically. At 80°C, similar rates of deamidation were observed for the disulfidebonded form, which is thermally unfolded, and the reduced form. At 37°C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30‐fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn‐67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the β‐turn of residues 66–68.
Structure and thermal stability of monomeric bacteriorhodopsin in mixed pospholipid/detergent micelles
Authors: Brouillette Christie G. , McMichens Ruth B. , Stern Lawrence J. , Khorana H. Gobind
Published: May 2005

Thermal unfolding experiments on bacteriorhodopsin in mixed Phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 ± 13 Å in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near‐UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation. We conclude that the tertiary structure of the bacteriorhodopsin monomer is essentially the same in micelles and the purple membrane. On the other hand, in the synthetic mixed micelle system, the packing between the nonnative amphiphiles and bacteriorhodopsin is probably not optimal, protein‐protein interactions have been lost, and thehelical packing may be looser because the crystalline lattice is absent. Itis likely that a combination of these effects leads to the decreased stability of micellar bacteriorhodopsin.
Prediction of the conformation of ice‐nucleation protein by conformational energy calculation
Authors: Mizuno Hiroshige
Published: May 2005

The active conformation of an icenucleation protein, whose major portion consists of a long polypeptide segment of nearly repetitive octapeptides, is predicted by the analyses of conformational energy and the mechanism of crystal growth. The protein ideally has an exact octapeptide repetition and is assumed to have a helical conformation. The present study searched for low‐energy helical conformations and each of the obtained low‐energy conformations examined as to whether it has a surface structure that can promote crystal formation. Two conformations obtained were good candidates for an ice nucleus. Both were found to have on their surfaces an arrangement of hydrogen‐bonding sites, which fits well with those of hydrogen bonds in hexagonal ice crystal. Further, one of the two conformations had a hexagonal conformational symmetry consistent with the hexagonal ice crystal structure. The other conformation had apentagonal conformational symmetry that could enable the growth of an ice crystal–dendritic polycrystalline snow crystal–which grows on metastable cubic ice.
Folding of bovine growth hormone is consistent with the molten globule hypothesis
Authors: Brems David N. , Havel Henry A.
Published: May 2005

Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two‐step folding mechanism. These results are summarized and interpreted according to the “molten globule” model. The molten globule state of bGH is characterized as a folding intermediate which largely a‐helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent‐exposed hydrophobic surface along helix 106127 that readily leads association.
Structural basis of hierarchical multiple substates of a protein. III: Side chain and main chain local conformations
Authors: Noguti Tosiyuki , Gō Nobuhiro
Published: May 2005

An analysis is carried out of differences in the minimum energy conformations obtained in the previous paper by energy minimization starting from conformations sampled by a Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatictrypsin inhibitor. Main conformational differences in each pair of energy minima are found usually localized in several side chains and in a few localmain chain segments. Such side chains and local main chain segments are found to take a few distinct local conformations in the minimum energy conformations. Energy minimum conformations can thus be described in terms of combinations of these multiple local conformations.
Structural basis of hierarchical multiple substates of a protein. IV: Rearrangements in atom packing and local deformations
Authors: Noguti Tosiyuki , Gō Nobuhiro
Published: May 2005

Differences in atom packing are studied in the minimum energy conformations derived from the record of the Monte Carlo simulation of conformational fluctuation in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations observed among the minima which are found in the previous paper are accompanied by rearrangement of atom packing. Spatial locations of the local deformations in the three‐dimensional folded structure are also studied. It is foundthat the local deformations are distributed in space in several clusters inthe folded structure. The size and location of the clusters characterize the respective fluctuations of the first and the second levels observed in the simulation. In the fluctuations of the first level local deformations, each of which usually involves a few side chains and one main chain local segment, are thermally exited independently of each other near thesurface of the molecule. The observed fluctuation of the second level involves a cooperative deformation involving many side chains and local main chain segments all in one cluster, which goes though the core of the molecule. The collective local deformations observed both in the first and second levels are plastic in the sense that they are accompanied with rearrangement of atom packing.
Structural basis of hierarchical multiple substates of a protein. V: Nonlocal deformations
Authors: Noguti Tosiyuki , Gō Nobuhiro
Published: May 2005

Distances between centers of gravity of individual residues are compared among the minimum energy conformations derived from the recordof the Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations originating from the multiplicity of localconformations cause deformations of the whole structure of the molecule in various ways, which can be classified into two types. Type 1:When a local deformation occurs in a region consisting of a few residues near the surfaceof the molecule, the whole shape of the molecule responds by deforming elastically. The magnitude of this deformation is in the range of thermalfluctuations calculated by the harmonic approximation around a singleminimum. Type 2: We have observed one case belonging to the second type in which local deformations occur cooperatively in an extended region. This regiongoes across the whole molecule and divide the remaining parts into two. Atom packing changes in and around the extended region of local deformations. For this reason deformation in this region is plastic. Relative locationand orientation between the divided two parts change very much. Deformationof the whole shape in this case, associated with the plastic deformationin an extended region, demonstrates that protein molecules have a flexibility beyond the harmonic limit.
Rebuilding flavodoxin from Cα coordinates: A test study
Authors: Reid Lorne S. , Thornton Janet M.
Published: May 2005

The tertiary structure of flavodoxin has been model build from only the X‐ray crystallographic α‐carbon coordinates. Main‐Chain atoms were generated from a dictionary of backbone structures. Side‐chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge‐based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 Å across all atoms to the native structure. Regular secondary structural elements were modeledmore accurately than other regions. About 40%of the ξ1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions.

The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contact, and hydrogen‐bonding patterns could identifypoorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.
Amino acid substitutions that increase the thermal stability of the λ Cro protein
Authors: Pakula Andrew A. , Sauer Robert T.
Published: May 2005

A mutant Cro protein, which bears the Ile‐30→ Leu substitution, is thermally unstable and degraded more rapidly than wildtype Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12°C to 14°C. These amino acid substitutions affect residues 16 and 26, which are substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild‐type Cro.
Size determination of multienzyme complexes using two‐dimensional agarose gel electrophoresis
Authors: Easom Richard A. , Debuysere Michael S. , Olson Merle S. , Serwer Philip
Published: May 2005

In the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two‐dimensional agarose gel electrophoresis (2d‐AGE) (Serwer, P. et al. Anal. Biochem. 152: 339–345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10–30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α‐ketoglutarate (α‐KGDC), and the branched chain α‐keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross‐linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d‐AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d‐AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.
Electrostatic effects in a large enzyme complex: Subunit interactions and electrostatic potential field of the icosahedral β60 capsid of heavy riboflavin synthase
Authors: Karshikov Andrej , Ladenstein Rudolf
Published: May 2005

The role of the electrostatic interactions in the stability of the icosahedral β60 capsid of heavy riboflavin synthase from Bacillus Subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interaction agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand‐free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between β‐subunits forming dimers, which connect the relatively stable pentamers in the β‐60 capsid. The release of the ligand causesareduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due tothe titratable groups and α‐helix macrodipoles has been calculated on the basic of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polardistribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of the positive potential “channels” that coincide with the channels constituted by the pentameric and trimeric β‐subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme‐substrate recognition.
A molecular dynamics analysis of protein structural elements
Authors: Post Carol Beth , Dobson Christopher M. , Karplus Martin
Published: May 2005

The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, β‐sheet, and loop structural elements in free and substrate‐bound hen egg‐white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and β‐sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of theloop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate‐bound simulation. A loop region (residues 101–107) of the active site cleft had significantly reduced motion due to interactions withthe substrate. Moreover, part of a loop and a 310 helix (residues of 67–88) not in contact with the substrate showeda marked increase in fluctuations. That these differences in dynamics of free and substrate‐bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuationsof the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain ϕ and ψ angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place eitherabruptly, in 1 psec or less, or with a diffusive character over 5–10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 Å rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface areaand the dynamics of the different structural elements.
Describing protein structure: A general algorithm yielding complete helicoidal parameters and a unique overall axis
Authors: Sklenar Heinz , Etchebest Catherine , Lavery Richard
Published: May 2005

We present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinated of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry. This algorithm has been implemented in a computer program named P‐Curve. Several examples of its possible applications are discussed.
Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm
Authors: Dohlman Jan G. , De Loof Hans , Prabhakaran M. , Koopman William J. , Segrest Jere P.
Published: May 2005

Eisenberg's helical hydrophobic moment (<μH>) algorithm was applied to the analysis of the primary structure of amphipathic α‐helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue <μH> for the amphipathic helical and control peptides were 0.46 (±/−0.07) and 0.33 (0.07) (P+0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular <μH> was plotted vs <μH>. The resulting curves are referred to as HM‐C. The mean HM‐C (of the two groups) was highly significantly different such that the HM‐C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic α helix at residues 4–21. This computer‐based method may enable rapid identification of peptide of the amphipathic α‐helix class.
Computational and site‐specific mutagenesis analyses of the asymmetric charge distribution on calmodulin
Authors: Weber P. C. , Lukas T. J. , Craig T. A. , Wilson E. , King M. M. , Kwiatkowski A. P. , Watterson D. Martin
Published: May 2005

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain α‐helices and calcium‐binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette‐based site‐specific mutagenesis of residues 82–84 or 118–120, the resulting proteins differ in their interaction with two distinct calmodulin‐dependent protein kinases, myosin light chain kinase and calmodulin‐ldependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charges in the region around residues 82–84 and 118–120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties that are important for the efficient assembly of calmodulin–protein kinase signal transduction complexes in eukaryotic cells.
Site‐directed mutagenesis of the T4 endonuclease V Gene: Mutations which enhance enzyme specific activity at low salt concentrations
Authors: Lloyd R. Stephen , Augustine Mary Lou
Published: May 2005

Previous structure/function analyses of the DNA repairenzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer‐specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832–1838 and 1839–1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has beeninvestigated by oligonucleotide site‐directed mutagenesis in which the codon for Tyr‐129 has been altered to reflect conservative changesof Trp and Phe and more dramatic changes of Ser‐ a stop, codon, deletion of the codon or introduction of a frameshift. Both changesto the aromatic amino acids resulted in proteins which accumulated will in E. coli and not only significantly enhanced the UV survival of repair, deficient cells but also complemented a defective denV gene within UV‐irradiated T4 phage. Partially purified preparations of the Tyr‐129 → Trp and Tyr‐129 → Phe mutants were assayed for their ability to processively incise UV‐irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr‐129 → Phe and Tyr‐129 → Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr‐129 → Phe mutant and to 20% that of wild type for the Tyr1‐29 → Trp mutant.
Hydrolysis of GTP by the α‐chain of Gs and other GTP binding proteins
Authors: Bourne Henry R. , Landis Claudia A. , Masters Susan B.
Published: May 2005

The functions of G proteins—like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)—depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP‐ and GDP‐bound states. Similarities in function and amino acid sequence indicate that EF‐Tu, p21ras, and G protein α‐chains evolved from a primordial GTP‐binding protein. Proteins in all three families appear to share common mechanisms for GTP‐dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the α‐chain of Gs (αs) point to key regions that are involved in GTP‐dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of αs in human pituitary tumors inhibit‐the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in αs that corresponds to Gln‐61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of the GTPase inhibiting mutations in αs occurs in the codon for an ARG residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by αs. This Arg residue is located in a domain of αs not represented in EF‐Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF‐Tu by the programmed ribosome and for p21ras by the recently discovered GTPase‐activating protein. Owing to their inherited similarities of structure and function, what we learn about αs, p21ras, or EF‐tu as individual molecules helps us to understand crucial functions of other members of the super‐family.
The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA
Authors: Beran‐Steed Rita K. , Tse‐Dinh Yuk‐Ching
Published: May 2005

Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA‐binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single‐stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C‐terminal domain was purified. It was eluted from the single‐stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno‐M13 DNA also demonstrated that the presence of the C‐terminal domain confers higher affinity to DNA for the enzyme.
A hydrophobic cluster forms early in the folding of dihydrofolate reductase
Authors: Garvey E. P. , Swank J. , Matthews C. R.
Published: May 2005

The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site‐directed mutagenesis and by physicochemical means. The absence of this fluorescence‐detected phase in the refolding of the Trp‐74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp‐74. In addition, water‐soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and nativelike conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp‐74 fromsolvent.
The interaction of calmodulin with fluorescent and photoreactive model peptides: Evidence for a short interdomain separation
Authors: O'Neil K. T. , DeGrado William F.
Published: May 2005

Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2+‐dependent manner. Recently, we introduced a photoaffinity label, p‐benzoylphenylalanine (Bpa), into the sequence of a model, α‐helical, calmodulin‐binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met‐144 on the C‐terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met‐71 on the N‐terminal do main was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long α‐helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin‐binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross‐link between Met‐71 and Met 124 on the N‐ and C‐terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthsized. After photocross‐linking the Bpa redidue of this peptide to Met‐71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C‐terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphi peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal sturcture of the uncommplexed protein.
The neuraminidase of influenza virus
Authors: Air Gillian M. , Laver W. Graeme
Published: May 2005

It is the enzyme neuraminidase, projecting form the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3‐dimensional structure of the epitopes was obtained by X‐ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.
Acid‐induced dimerization of skeletal troponin C
Authors: Wang Chien‐Kao , Lebowitz Jacob , Cheung Herbert C.
Published: May 2005

We have investigated pH‐dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low‐speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20°C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20°C, and was completely prevented at a rotor speed of 40,000 rpm and 4°C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met‐25 and Cys‐98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH‐dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The protein‐induced increase in the energy transfer distance is related to the acid‐induced dimerization of the protein. The extended conformation observed at pH 5.2 is compatible with the dumbbell‐shaped structure of skeletal troponin C crystals obtained from turkey at pH 5.0 [Herzberg, O., James, M. N. G. Nature (London) 313:653–659, 1985] and chicken at pH 5.1 (Sundaralingam, M., Bergstrome, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., Wang, B. C. Science 227:945–948, 1985). However, the conformation in neural solution deviates form that predicted by crystallography. Intermolecular interactions leading to dimer formation likely play an important role in promoting the extended conformation that exists at acidic pH.