bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of the molecular basis for β‐adrenergic receptor subtype specificity
Authors: Dixon Richard A. F. , Hill Wendy S. , Candelore Mari R. , Rands Elaine , Diehl Ronald E. , Marshall Mark S. , Sigal Irving S. , Strader Catherine D.
Published: May 2005

Pharmacological analysis of ligand binding to the β‐adrenergic receptor (βAR) has revealed the existence of two distinct receptor subtypes (β1 and β2) which are the products of different genes. The predicted amino acid sequence of the β1 and β2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human β1 and hamster β2 receptors. Analyses of the ligand‐binding characteristics of these hybrid receptors revealed that residues in the middle portion of the βAR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacement of regions of the hamster β2AR with the analogous regions from the avian β1AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand‐binding properties of the receptor, the subtype‐specific differences in amine‐substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the β1 and β2 receptors.
Chemical modification of the RTEM‐1 thiol β‐lactamase by thiol‐selective reagents: Evidence for activation of the primary nucleophile of the β‐lactamase active site by adjacent functional groups
Authors: Knap Anna K. , Pratt R. F.
Published: May 2005

The RTEM‐1 thiol β‐lactamase (Sigal, I. S., Harwood, B. G., Arentzen, R., Proc. Natl. Acad. Sci. USA. 79:7157–7160, 1982) is inactivated by thiol‐selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4′‐dipyridyldisulfide, which modify the active site thiol group. The pH‐rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol β‐lactamase is stabilized by two hydrogen‐bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys‐73 ammonium group, of pKa around 6.7, is less clear, but may be the conserved Glu‐166 carboxylic acid. β‐Lactamase activity is associated with the EH2 form, and thus the β‐lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol β‐lactamase) and two acidic (hydrogen‐bond donor) groups (one of which is likely to be the above‐mentioned lysine ammonium group).
Use of restrained molecular dynamics in water to determine three‐dimensional protein structure: Prediction of the three‐dimensional structure of Ecballium elaterium trypsin inhibitor II
Authors: Chiche Laurent , Gaboriaud Christine , Heitz Annie , Mornon Jean‐Paul , Castro Bertrand , Kollman Peter A.
Published: May 2005

Refinement of distance geometry (DG) structures of EETI‐II (Heitz et al.:Biochemistry 28:2392–2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined “in cacuo.” The consistent lower values of residual NMR constraint violations, apolar/polar surface ration and solvation free energy of one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI‐II. Except for the few first residues for which no NMR constraints were available, this computer model fully agree with X‐ray structures of CMTI‐I (Boe et al.: FEBS Lett. 242:285–292, 1989) and EETI‐II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics n water is thus proved to highly valuable for refinement of DG structures Also, the successful use of apolar/polar surface ratio and solvation free energy reinforce the analysis of Novotny et al. (Proteins 4: 19–30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.
Role of phosphorylation in conformational adaptability of bovine myelin basic protein
Authors: Deibler Gladys E. , Stone Audrey L. , Kies Marian W.
Published: May 2005

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated forms(s) of component1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phophorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in β‐structure(s).
Apolar peptide models for conformational heterogeneity, hydration, and packing of polypeptide helices: Crystal structure of hepta‐ and octapeptides containing α‐aminoisobutyric acid
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Uma K. , Balaram P.
Published: May 2005

The crystal structures of two helical peptides Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Leu‐OMe (VALU‐7) and Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Leu‐Aib‐OMe (VALU‐8) have been determined to a resolution of 1.0 and 0.9 Å, respectively. Both the seven and eight residue peptides crystallize with two conformers per asymmetric unit. The VALU‐8 conformers are completely helical and differ only at the C‐terminus by a sign reversal of the ϕ, ψ angles of the last residue. One of the VALUE‐7 conformers occurs as a normal α‐helix, whereas in the other, the N(7)O(3) α‐type hydrogen bond is ruptured by the entry of a water molecule (W) into the helix, which in turn makes hydrogen bonds N(7) ⃛W = 2.97 Å and ⃛O(3) = 2.77 Å. The other side of the water molecule is surrounded by a hydrophobic pocket. These two conformers give a static representation of a step in a possible helix unwinding or folding process. In the value‐8 crystal the helices aggregate in a parallel mode, whereas the aggregation is antiparallel in the VALU‐7 crystal. The crystal parameters are VALUE‐7 crystal. The crystal parameters are VALUE‐7, P21, a = 10.203 (3) Å, b = 19.744 (6) Å, c = 22.561 (6) Å, α = 96.76°, Z = 4, C38, H69N7O10·0.5 H2O, R = 6.65% for 3674 reflections observed >3σ(F): and VALU‐8, P21, a; = 10.596 (4) Å, b = 27.57 (6) Å, c = 17.745 (5) Å, β = 95.76 (3)°, Z = 4, C42H76N76O11·0.25 CH3OH, R = 6.63% for 4701 reflections observed >3σ(F).
Evaluation of homology modeling of HIV Protease
Authors: Weber Irene T.
Published: May 2005

The model of human immuno‐deficiency virus (HIV‐1) protease which was based on the crystal structure of Rous sarcoma virus (RSV) protease has been compared to the recently determined crystal structure of chemically synthesized HIV‐1 protease. The overall difference between the model and crystal structure was 1.4 Å root mean square (rms) deviation for 86 superimposed Cα atoms. The position of the flexible flap differs in the model and six residues at the amino terminus were incorrectly placed. With these exceptions, all atoms of the model and crystal structure agree to 2.11 Å runs deviation. The conformation of some surface bends in the model agrees less well with the crystal structure. Identical amino acids in RSV and HIV proteases were modeled more reliably than different types of amino acids. The amino acids which form the substrate binding site were modeled most accurately to 1.2 Å rms deviation for all atoms compared to the crystal structure. This suggests that functionally significant regions of related proteins can be modeled with high accuracy. The model gave correct predictions for residues making interactions with the substrate, and thereof could be used to design inhibitors, The model based on the RSV protease structure is more similar to the experimental structure than are previous models based on the structures of non‐viral aspartic proteases.
Solution structure of a zinc finger domain of yeast ADR1
Authors: Klevit Rachel E. , Herriott Jon R. , Horvarth Suzanna J.
Published: May 2005

The “zinc finger” is a 30‐residue repeating motif that has been identified in a variety of eukaryotic transcription factors. Each domain is capable of binding a Zn2+ ion through invariant Cys and His residues. We have determined the three‐dimensional structure of a synthetic peptide that corresponds to one of the two zinc finger domains in the yeast transcription factor ADR1, using two‐dimensional nuclear magnetic resonance spectroscopy. The Zn2+ ‐bound structure of the peptide consists of a loop containing the two Cys residues, a “fingertip,” a 12‐ to 13‐residue α‐helix containing the two His residues, and a C‐terminal tail. A majority of the interrresidue contacts observed invlove the seven conserved residues of the prototypic zinc finger (i.e., four zinc ligands and the three hydrophobic residues), indicating that these residues are largely responsible for the three‐dimensional structure of the domain and that all the zinc finger domains of the TFIIIA class will have similar structures, with the highest concentration of such residues on the external face of the α‐helix.
ALMA, an editor for large sequence alignments
Authors: Thirup Soren , Larsen Niels E.
Published: May 2005

A dedicated sequence editor ALMA, was developed for aligning many sequences of proteins or RNA molecules or longer DNA fragments. Like previously published editors, ALMA is menu directed, screen oriented, and offers similarity and consensus display. ALMA has the additional features of collective movement of sequences, acceptance of input from many sources including structure files and databases, secondary structure display, and easy merging of alignments. In order to maintain sequence integrity and save disk space, gaps and sequences are stored separately. Automatic recovery of a session is possible. Finally, The program allows interaction between manual and automatic alignment.
Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis
Authors: Henrissat Bernard , Saloheimo Markku , Lavaitte Stéphane , Knowles Jonathan K. C.
Published: May 2005

A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.
Normal mode analysis of human lysozyme: Study of the relative motion of the two domains and characterization of the harmonic motion
Authors: Gibrat Jean‐François , Gō Nobuhiro
Published: May 2005

A normal mode analysis of human lysozyme has been carried out at room temperature. Human lysozyme is an enzyme constituted of two domains separated by an active site cleft, the motion of which is thought to be relevant for biological function. This motion has been described as a hinge bending motion. McCammon et al.1 have determined the characteristics of the hinge bending motion but they assumed a prior knowledge of the hinge axis. In this work we propose a method which is free from this assumption and determines the hinge axis and root mean square (rms) rotation angle which give the best agreement with the pattern of changes in all the distances between nonhydrogen atoms in the two domains, obtained by the normal mode analysis. The hinge axis we found is notably different from the one previously determined and goes, roughly, through the Cα 55 and Cα 76, i.e., it is located at the base of the β‐sheet of the second domain. The rms value for the rotation angle is also twice as large as the previous one: 3.37°. It is shown that this hinge bending motion provides a fairly good approximation of the dynamics of human lysozyme and that the normal mode with the lowest frequency has a dominating contribution to this hinge bending motion.

A study of the accessible surface area of the residues within the cleft reveals that the motion does not result in a better exposure to the solvent of these residues.

A characterization of the thermally excited state (under the hypothesis of the harmonicity of the potential energy surface) has been done using the concept of topology of atom packing. Under this hypothesis the thermal fluctuations result only in a small change of the topology of atom packing, leading therefore to nearly elastic deformations of the protein.
Design of biologically active, conformationally constrained GnRH antagonists
Authors: Struthers R. Scott , Tanaka Genzo , Koerber Steven C. , Solmajer Tom , Baniak Edmund L. , Gierasch Lila M. , Vale Wylie , Rivier Jean , Hagler Arnold T.
Published: May 2005

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin‐releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[Δ3,4 Pro‐D4ClPhe‐DTrp‐Ser‐Tyr‐DTrp‐NMeLeu‐Arg‐Pro‐βAla], led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two‐dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain–side chain linkages suggested by the conformational model:
These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure–activity studies.
Hydrogen bonds involving sulfur atoms in proteins
Authors: Gregoret Lydia M. , Rader Stephen D. , Fletterick Robert J. , Cohen Fred E.
Published: May 2005

Intrachain hydrogen bonds are a hallmark of globular proteins. Traditionally, these involve oxygen and nitrogen atoms. The electronic structure of sulfur is compatible with hydrogen bond formation as well. We surveyed a set of 85 high‐resolution protein structures in order to evaluate the prevalence and geometry of sulfur‐containing hydrogen bonds. This information should be of interest to experimentalists and theoreticians intersted in protein structure and protein engineering.
The frequency of ion‐pair substructures in proteins is quantitatively related to electrostatic potential: A statistical model for nonbonded interactions
Authors: Bryant Stephen H. , Lawrence Charles E.
Published: May 2005

A statistical analysis of ion pairs in protein crystal structures shows that their abundance with respect to uncharged controls is accurately predicted by a Botlzmann‐like function of electrostatic potential. It appears that the mechanisms of protein folding and/or evolution combine to produce a “thermal” distribution of local nonbonded interactions, as has been suggested by statistical–mechanical theories. Using this relationship, we develop a maximum likelihood methodology for estimation of apparent energetic parameters from the data base of known structures, and we derive electrostatic potential functions that lead to optimal agreement of observed and predicted ion‐pair frequencies. These are similar to potentials of mean force derived from electrostatic theory, but departure from Coulombic behavior is less than has been suggested.
Single‐stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids
Authors: Ruvolo Peter P. , Keating Kathleen M. , Williams Kenneth R. , Chase John W.
Published: May 2005

The DNA and protein sequences of single‐stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F‐plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2‐terminal two‐thirds of the protein, they diverge in the COOH‐terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp‐40, Trp‐54, Trp‐88, His‐55, and Phe‐60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr‐70 in DNA binding is suggested for the first time. Although the COOH‐terminal third of these proteins diverges more than their NH2‐terminal regions, the COOH‐terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2‐terminal core and an acidic COOH‐terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F‐plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A‐T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix‐destabilizing proteins than is the E. coli SSB protein.
Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths
Authors: Bell Jeffrey A. , Wilson Keith P. , Zhang Xue‐Jun , Faber Howard R. , Nicholson Hale , Matthews Brian W.
Published: May 2005

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X‐ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X‐ray diffraction data set.

The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2‐mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg‐52 and the disruption of the salt bridge between this residue and Glu‐62.

Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.
Computational studies of ligand diffusion in globins: I. Leghemoglobin
Authors: Czerminski Ryszard , Elber Ron
Published: May 2005

The thermally assisted diffusion of a small ligand (carbon monoxide) through a protein matrix (lupine leghemoglobin) is investigated computationally. The diffusion paths are calculated by a varient of the time‐dependent Hartree approximation which we call LES (locally enhanced sampling). The variant which was recently introduced by Elber and Karplus1 is based on the classical TD‐SCF approximation of Gerber et al.2 The simulation enables more significant search for diffusion pathways than was possible before. This is done by increasing the number of ligand trajectories using a single trajectory for the protein. We compare qualitatively diffusion rates in leghemoglobin and in myoglobin. The calculation shows that the diffusion in leghemoglobin is much faster than the diffusion in myoglobin, in agreement with experiment. The gate in leghemoglobin is opened by fluctuations at a close contact between the B/C and the G helices. The most relevant fluctuation is the rigid shift of the C helix with respect to the G helix. This path is not observed in a comparable calculation for myoglobin.1 This finding is rationalized by the lack of the D helix in leghemoglobin and a significantly more flexible CE loop. Supporting experimental evidence for the importance of the CE loop in leghemoglobin can be found in the kinetics studies of Gibson et al.28
Side chain–backbone hydrogen bonding contributes to helix stability in peptides derived from an α‐helical region of carboxypeptidase A
Authors: Bruch Martha D. , Dhingra Madan M. , Gierasch Lila M.
Published: May 2005

Recently, Presta and Rose proposed1 that a necessary condition for helix formation is the presence of residues at the N‐and C‐termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171–188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration‐independent CD parameters which are sensitive probes of helix formation.
Cu(II)‐Binding properties of a cytochrome c with a synthetic metal‐binding site: His‐X3‐His in an α‐helix
Authors: Todd Robert J. , Van Dam Mariana E. , Casimiro Danilo , Haymore Barry L. , Arnold Frances H.
Published: May 2005

A metal‐binding site consisting of two histidines positioned His‐X3‐His in an α‐helix has been engineered into the surface of Saccharomyces cerevisiae iso‐1‐cytochrome c. The synthetic metal‐binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two‐phase polymer systems containing a polymer‐metal complex, Cu(II)IDA‐PEG, and by metal‐affinity chromatography. The stability constant for the complex formed between Cu(II)IDA‐PEG and the cytochrome c His‐X3‐His site is 5.3 × 104 M−1, which corresponds to a chelate effect that contributes 1.5 kcal mol−1 to the binding energy. Incorporation of the His‐X3‐His site yields a synthetic metal‐binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.
Thermodynamics of ligand binding and denaturation for his64 mutants of tissue plasminogen activator kringle‐2 domain
Authors: Kelley Robert F. , DeVos Abraham M. , Cleary Scott
Published: May 2005

The contribution of His64 to the function and stability of tissue plasminogen activator (t‐PA) kringle‐2 domain (His244 in t‐PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild‐type kringle‐2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies1 suggested that the His64 side chain could play an important role in ligand binding by forming an ion‐pair with the carboxylate of the ligand, L‐lysine. Kringle‐2 domains were expressed by secretion of the 174‐263 portion of t‐PA in E. coli and purified as previously described for the wild‐type domain.2 Both mutant proteins retain affinity for L‐lysine, although reduced three‐ to four‐fold relative to wild‐type, demonstrating that His64 does not interact with the ligand carboxylate through an ion‐pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 Å chain length, whereas the wild‐type domain prefers an 8.8 Å long ligand. For both wild‐type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (ΔH = −6,000 to −7,000 cal/mol) and TΔS accounts for < 15% of ΔG. In addition, the H64Y mutant differs from wild‐type in the effect of ligand α‐amino group modification on binding affinity. Based on examination of the x‐ray structure recently determined for wild‐type kringle‐2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild‐type protein with the difference in stabilization free energy (ΔΔG) equal to 2.7 kcal/mol at 25°C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.
Experimental and theoretical studies of the three‐dimensional structure of human interleukin‐4
Authors: Curtis Benson M. , Presnell Scott R. , Srinivasan Subhashini , Sassenfeld Helmut , Klinke Ralph , Jeffery Eric , Cosman David , March Carl J. , Cohen Fred E.
Published: May 2005

The structure of human interleukin 4 (IL‐4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL‐4 belonged to the all α‐helix class of protein structures. Secondary structure prediction, site‐directed mutagenesis, and CD spectroscopy suggested a predominantly α‐helical structure, consistent with a four‐helix bundle structural motif. A human/mouse IL‐4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL‐4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four‐helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.
Thiol proteases and aldehyde dehydrogenases: Evolution from a common thiolesterase precursor?
Authors: Hempel John , Nicholas Hugh , Jörnvall Hans
Published: May 2005

The C‐terminal 222 residues of human liver aldehyde dehydrogenase can be aligned with the C‐terminal 226 residues of a thiol protease from Dictyostelium discoideum to yield 47 residue identities, including matching active site cysteine residues. A multiple alignment with three more aldehyde dehydrogenases and three more thiol proteases yields three regions with clustered residue similarities. In the tertiary structure of papain, these three regions are in close proximity although widely separated in primary structure, and many conserved residues are located in the active site groove. The three‐dimensional relationships, the common thiol ester mechanisms of the enzymes, the locations of exon boundaries in the dehydrogenase and protease genes, and the conservation of internal salt‐bridging and disulfide‐paired residues in papain, all appear compatible with the hypothesis of an ancestral relationship between thiol proteases and aldehyde dehydrogenases.
FASTRUN: A special purpose, hardwired computer for molecular simulation
Authors: Fine Richard , Dimmler Gerd , Levinthal Cyrus
Published: May 2005

We describe the design, construction, and performance of a special purpose, hardwired accelerator for molecular mechanical calculations called FASTRUN. The processor was designed at Columbia University in 1984, constructed in the Instrumentation Division of Brookhaven National Laboratory, and delivered to Columbia in final form in 1989. It was rendered functional for molecular mechanics in early 1990. Together with its host Star array processor, FASTRUN has a measured performance for molecular dynamics simulations which compares favorably with present day supercomputers. The hardware replication cost of FASTRUN is on the order of $100,000.00
Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons
Authors: Nicholls Anthony , Sharp Kim A. , Honig Barry
Published: May 2005

We demonstrate in this work that the surface tension, water‐organic solvent, transfer‐free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well‐known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25°C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.
Computer design of bioactive molecules: A method for receptor‐based de novo ligand design
Authors: Moon Joseph B. , Howe W. Jeffrey
Published: May 2005

The design of molecules to bind specifically to protein receptors has long been a goal of computer‐assisted molecular design. Given detailed structural knowledge of the target receptor, it should be possible to construct a model of a potential ligand, by algorithmic connection of small molecular fragments, that will exhibit the desired structural and electrostatic complementarity with the receptor. However, progress in this area of receptorbased, de novo ligand design has been hampered by the complexity of the construction process, in which potentially huge numbers of structures must be considered. By limiting the scope of the structure‐space examined to one particular class of ligands–namely, peptides and peptide‐like compounds–the problem complexity has been reduced to the point that successful, de novo design is now possible. The methodology presented employs a large template set of amino acid conformations which are iteratively pieced together in a model of the target receptor. Each stage of ligand growth is evaluated according to a molecular mechanicsbased energy function, which considers van der Waals and coulombic interactions, internal strain energy of the lengtheining ligand, and desolvation of both ligand and receptor. The search space is managed by use of a data tree which is kept under control by pruning according to the energy evaluation. Ligands grown by this procedure are subjected to follow‐up evaluation in which an approximate binding enthalpy is determined. This methodology has proven useful as a precise model‐builder and has also shown the ability to design bioactive ligands.
Subunit assembly and active site location in the structure of glutamate dehydrogenase
Authors: Baker Patrick J. , Britton K. Linda , Engel Paul C. , Farrants George W. , Lilley Kathryn S. , Rice David W. , Stillman Timothy J.
Published: May 2005

The three‐dimensional crystal structure of the NAD+‐linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 Å resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self‐assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2‐oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.
Protein design on computers. Five new proteins: Shpilka, grendel, fingerclasp, leather, and aida
Authors: Sander Chris , Vriend Gerrit , Bazan Fernando , Horovitz Amnon , Nakamura Haruki , Ribas Luis , Finkelstein Alexei V. , Lockhart Andrew , Merkl Rainer , Perry L. Jeanne , Emery Stephen C. , Gaboriaud Christine , Marks Cara , Moult John , Verlinde Christophe , Eberhard Marc , Elofsson Arne , Hubbard Tim J. P. , Regan Lynne , Banks Jay , Jappelli Roberto , Lesk Arthur M. , Tramontano Anna
Published: May 2005

What is the current state of the art in protein design? This question was approached in a recent two‐week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four‐stranded β‐sheets, a scaffold on which to explore variations in loop topology; Grendel, a four‐helical membrane anchor, ready for fusion to water‐soluble functional domains; Fingerclasp, a dimer of interdigitating β–β–α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather—a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three‐dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.
Domain flexibility in aspartic proteinases
Authors: Šali Andrej , Veerapandian B. , Cooper Jon B. , Moss David S. , Hofmann Theo , Blundell Tom L.
Published: May 2005

Comparison of the three‐dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X‐ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190–302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports and model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.
Deconvolution of the circular dichroism spectra of proteins: The circular dichroism spectra of the antiparallel β‐sheet in proteins
Authors: Perczel A. , Park K. , Fasman Gerald D.
Published: May 2005

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure β‐pleated sheet in globular proteins. On the basis of X‐ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:‐669–679, 1991), was improved by the addition of proteins with high β‐pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (α‐helix, antiparallel β‐pleated sheet, β‐turns, and unordered conformation), as well as a curve attributed to the “aromatic contribution” in the wavelength range of 195–240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X‐ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X‐ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the α‐helical structure, but upon analysis yielded a considerable amount of β‐sheet in agreement with the X‐ray structure. © 1992 Wiley‐Liss, Inc.
Mutating the charged residues in the binding pocket of cellular retinoic acid‐binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability
Authors: Zhang Jianhua , Liu Zhi‐Ping , Jones T. Alwyn , Gierasch Lila M. , Sambrook Joseph F.
Published: May 2005

Three‐dimensional modeling of the complex between retinoic acid‐binding protein (CRABP) and retinoic acid suggests thatbinding of the ligand is mediated by interaction between the carboxyl groupof retinoic acid and two charged amino acids (Arg‐111 and Arg‐131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein–ligand interaction, amino acid substitutions were made by oligonucleotide‐directed, site‐specific mutagenesis. The wild‐type and mutant proteins were expressed in E. coli and subsequently purified. Like wild‐type CRABP, the mutant proteins are composed mainly of β‐strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild‐type protein. Mutants in which Arg‐111 and Arg‐131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild‐type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild‐type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP–retinoic acid complex. These data suggest a model for CRABP–retinoic acid interaction in which the repulsive forces between the positively‐charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively‐charged pair of amino acids produces a protein that is more thermostable than wild‐type CRABP but less effective at ligand‐binding. © 1992 Wiley‐Liss, Inc.
Continuum electrostatics of the C‐peptide: Anatomy of the problem
Authors: Rashin Alexander A.
Published: May 2005

A computational study of the role of all ionizable groups of the C‐peptide in its helix–coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix–coil equilibrium of the C‐peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix–coil transition due to the side chains of the C‐peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix–coil transition of the C‐peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed. © 1992 Wiley‐Liss, Inc.
Michaelis complexes of porcine pancreatic elastase with 7‐[(alkylcarbamoyl)amino]‐4‐chloro‐3‐ethoxyisocoumarins: Translational sampling of inhibitor position and kinetic measurements
Authors: Plaskon R. Richard , Kam Chih‐Min , Burgess Edward M. , Powers James C. , Suddath F. L.
Published: May 2005

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7‐[(alkylcarbamoyl)amino]‐4‐chloro‐3‐ethoxyisocoumarins (alkylHNCO‐EICs) is the formation of the non‐covalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO‐EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO‐EIC inhibitors of PPE. Two novel alkylHNCO‐EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed. © 1992 Wiley‐Liss, Inc.
Crystallization and preliminary X‐ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus
Authors: Huang Kui , Kodandapani R. , Kallwass Helmut , Hogan James K. , Parris Wendy , Friesen James D. , Gold Marvin , Jones J. Bryan , James Michael N. G.
Published: May 2005

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three‐dimensional structure solved, the gene coding for it has been cloned, and the proteincan be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4in 50 mM piperazine HCI buffer. The crystals belong to space group P6622 (P6622) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74–92, 1990) crystallized in the space group (P61) with a = 102.3 Å, c = 168.6 Å for the R171W, Q102R, C97G triple mutant, and a = 98.2 Å; c = 162.1 Å for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one‐quarter of a tetramer (Mr 135,000)in the asymmetric unit and have (VM values of 3.8 and 3.3 Å3/dalton, respectively). The R171W mutant diffracts to 2.5 Å and the R171Y mutant to approximately 3.5 Å © 1992 Wiley‐Liss, Inc.