bluuebunny/crossref_metadata_2025 huggingface dataset:
Volume changes of the molten globule transitions of horse heart ferricytochrome c: A thermodynamic cycle
Authors: Foygel Kira , Spector Shari , Chatterjee Sukalyan , Kahn Peter C.
Published: February 2009

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be —136 ± 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded +100 ± 6 mL/mol. MG to N was broken into two steps, N to NClX at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClX = N interacting with an unknown number, X, of chloride ions), and MG to NClX by jumping MG at pH 2 in 0.5 M KCl to pH 7 at the same salt concentration. The ΔV of N to NClX was —30.9 ± 1.4 mL/mol protein, whereas MG to NClX entailed a ΔV of —235 ± 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.
Structural analysis of the N‐ and C‐termini in a peptide with consensus sequence
Authors: Gong Youxiang , Zhou Hongxing X. , Guo Mingming , Kallenbach Neville R.
Published: February 2009

We present a structural analysis of a peptide, the sequence of which includes amino acids that show preferences for specific positions near the N‐ and C‐termini in protein helices. This peptide has the sequence ac‐YMSEDELKAAEAAFKRHGVP‐amide, which includes a strong version of an N‐terminal Harper‐Rose capping box structure as well as a Gly located close to the C‐terminus designed to elucidate its role in C‐terminal capping. The sequence of five residues at the middle is inserted to separate effects at the two ends via a helix‐stabilizing linker. Application of a simulated annealing procedure using interproton distance constraints derived from 1H NOESY experiments in water reveals the presence of a C‐terminal structure in this model. The C‐terminus forms a folded back structure in a significant fraction of structures generated by the annealing, in most of which Gly assumes an αL conformation. This structure occurs within a highly flexible region of the molecule and hence is occupied only a fraction of the time.
Hints on the evolutionary design of a dimeric RNase with special bioactions
Authors: Donato Alberto Di , Cafaro Valeria , Romeo Ivana , D'Alessio Giuseppe
Published: February 2009

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394–17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N‐terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N‐terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N‐teminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.
Structural energetics of barstar studied by differential scanning microcalorimetry
Authors: Wintrode Patrick L. , Griko Yuri V. , Privalov Peter L.
Published: February 2009

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two‐state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at ≈︁75 °C was found to be 5.6 ± 0.3 kJ K−1 mol−1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 ± 0.03 cal K−1 g−1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase. Our data, which indicate that barstar has a loosely packed interior, suggest that high conformational flexibility of barstar's native structure may play an important role in allowing it to optimize its contacts with barnase upon binding without disrupting favorable, tightly packed internal interactions.
Urea‐induced dissociation and unfolding of dodecameric glutamine synthetase from escherichia coli: Calorimetric and spectral studies
Authors: Zolkiewski Michal , Nosworthy Neil J. , Ginsburg Ann
Published: February 2009

Urea‐induced dissociation and unfolding of manganese · glutamine synthetase (Mn‐GS) have been studied at 37 °C (pH 7) by spectroscopic and calorimetric methods. In 0 to ∼2 M urea, Mn‐GS retains its dodecameric structure and full catalytic activity. Mn‐GS is dissociated into subunits in 6 M urea, as evidenced by a 12‐fold decrease in 90° light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra‐ring and inter‐ring subunit contacts, respectively, in the layered hexagonal structure of Mn‐GS. The enthalpies for transferring Mn‐GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn · GS is 14 ± 4 cal/g. A net proton uptake of ∼50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn‐GS and the number of binding sites increasing ∼9‐fold during the protein unfolding.
Finding flexible patterns in unaligned protein sequences
Authors: Jonassen Inge , Collins John F. , Higgins Desmond G.
Published: February 2009

We present a new method for the identification of conserved patterns in a set of unaligned related protein sequences. It is able to discover patterns of a quite general form, allowing for both ambiguous positions and for variable length wildcard regions. It allows the user to define a class of patterns (e.g., the degree of ambiguity allowed and the length and number of gaps), and the method is then guaranteed to find the conserved patterns in this class scoring highest according to a significance measure defined. Identified patterns may be refined using one of two new algorithms. We present a new (nonstatistical) significance measure for flexible patterns. The method is shown to recover known motifs for PROSITE families and is also applied to some recently described families from the literature.
Multidomain organization of eukaryotic guanine nucleotide exchange translation initiation factor eIF‐2B subunits revealed by analysis of conserved sequence motifs
Authors: Koonin Eugene V.
Published: February 2009

Computer‐assisted analysis of amino acid sequences using methods for database screening with individual sequences and with multiple alignment blocks reveals a complex multidomain organization of yeast proteins GCD6 and GCD1, and mammalian homolog of GCD6 — subunits of the eukaryotic translation initiation factor eIF‐2B involved in GDP/GTP exchange on eIF‐2. It is shown that these proteins contain a putative nucleotide‐binding domain related to a variety of nucleotidyltransferases, most of which are involved in nucleoside diphosphate‐sugar formation in bacteria. Three conserved motifs, one of which appears to be a variant of the phosphate‐binding site (P‐loop) and another that may be considered a specific version of the Mg2+‐binding site of NTP‐utilizing enzymes, were identified in the nucleotidyltransferase‐related domain. Together with the third unique motif adjacent to the P‐loop, these motifs comprise the signature of a new superfamily of nucleotide‐binding domains. A domain consisting of hexapeptide amino acid repeats with a periodic distribution of bulky hydrophobic residues (isoleucine patch), which previously have been identified in bacterial acetyltransferases, is located toward the C‐terminus from the nucleotidyltransferase‐related domain. Finally, at the very C‐termini of GCD6, eIF‐2Be, and two other eukaryotic translation initiation factors, eIF‐4γ and eIF‐5, there is a previously undetected, conserved domain. It is hypothesized that the nucleotidyltransferase‐related domain is directly involved in the GDP/GTP exchange, whereas the C‐terminal conserved domain may be involved in the interaction of eIF‐2B, eIF‐4γ, and eIF‐5 with eIF‐2.
Molecular model of the N‐terminal receptor‐binding domain of the human CD6 ligand ALCAM
Authors: Bajorath Jürgen , Bowen Michael A. , Aruffo Alejandro
Published: February 2009

CD6‐ligand interactions have been implicated in the regulation of T‐cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin‐like domains. As a fusion protein, the N‐terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR‐analogous loops, the location of N‐linked glycosylation sites, and residues that form the β‐sheet faces of the immunoglobulin‐like domain. Predicted structural characteristics of the A'GFCC “C” face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor‐ligand interactions.
Crystallization of the chaperone protein SecB
Authors: Vrielink Alice , Beamer Lesa , Le Thanh , Eisenberg David
Published: February 2009

The secretory protein SecB found inEscherichia coliis a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space. SecB maintains these proteins in a translocation‐competent conformation facilitating the translocation process. The material has been cloned and expressed inE. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensionsa= 56.0 Å,b= 111.1 Å,c= 134.7 Å, and β = 104°. The crystals diffract to 8 Å resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.
Structural analysis of zinc substitutions in the active site of thermolysin
Authors: Holland Debra R. , Hausrath Andrew C. , Juers Doug , Matthews Brian W.
Published: February 2009

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active‐site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601–4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal‐substituted thermolysins at 1.7–1.9 Å resolution.

The structure of the Co2+‐substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt‐relative to the zinc‐substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+‐ and Mn2+‐substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side‐chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side‐chain movements are similar to adjustments that have been observed previously in association with the “hinge‐bending” motion that is presumed to occur during catalysis by the zinc endoproteases.

In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 Å of the zinc bound to native thermolysin, explaining the inhibitory effect.
Stromelysin‐1: Three‐dimensional structure of the inhibited catalytic domain and of the C‐truncated proenzyme
Authors: Becker Joseph W. , Marcy Alice I. , Rokosz Laura L. , Axel Melinda G. , Burbaum Jonathan J. , Fitzgerald Paula M.D. , Cameron Patricia M. , Esser Craig K. , Hagmann William K. , Hermes Jeffrey D. , Springer James P.
Published: February 2009

The proteolytic enzyme stromelysin‐1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin‐1 is synthesized as a proenzyme that is activated by removal of an N‐terminal prodomain. The active enzyme contains a catalytic domain and a C‐terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three‐dimensional structures of both a C‐truncated form of the proenzyme and an inhibited complex of the catalytic domain by X‐ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three α‐helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino‐to‐carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
Water molecules participate in proteinase‐inhibitor interactions: Crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B
Authors: Huang Kui , James Michael N.G. , Lu Wuyuan , Laskowski Michael , Anderson Stephen
Published: February 2009

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water‐mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, Ahx18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol−1 Å−2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site‐directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein‐complex stability.
Thermal stability determinants of chicken egg‐white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules
Authors: Shih Phoebe , Kirsch Jack F. , Holland Debra R.
Published: February 2009

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 °C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40‐Ile 55‐Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side‐chain volume (r = 0.75). The X‐ray structures of the S91A (1.9 Å) and I55L/S91T/D101S (1.7 Å) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to ΔΔG298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.
Atomic solvation parameters in the analysis of protein‐protein docking results
Authors: Cummings Maxwell D. , Read Randy J. , Hart Trevor N.
Published: February 2009

Several sets of amino acid surface areas and transfer free energies were used to derive a total of nine sets of atomic solvation parameters (ASPs). We tested the accuracy of each of these sets of parameters in predicting the experimentally determined transfer free energies of the amino acid derivatives from which the parameters were derived. In all cases, the calculated and experimental values correlated well. We then chose three parameter sets and examined the effect of adding an energetic correction for desolvation based on these three parameter sets to the simple potential function used in our multiple start Monte Carlo docking method. A variety of protein‐protein interactions and docking results were examined. In the docking simulations studied, the desolvation correction was only applied during the final energy calculation of each simulation. For most of the docking results we analyzed, the use of an octanol‐water‐based ASP set marginally improved the energetic ranking of the low‐energy dockings, whereas the other ASP sets we tested disturbed the ranking of the low‐energy dockings in many of the same systems. We also examined the correlation between the experimental free energies of association and our calculated interaction energies for a series of proteinase‐inhibitor complexes. Again, the octanol‐water‐based ASP set was compatible with our standard potential function, whereas ASP sets derived from other solvent systems were not.
Thrombin‐binding affinities of different disulfide‐bonded isomers of the fifth EGF‐like domain of thrombomodulin
Authors: Hunter Michael J. , Komives Elizabeth A.
Published: February 2009

The fifth EGF‐like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide‐bonded isomers. The domain without the connecting region formed three disulfide‐bonded isomers upon refolding under redox conditions. Of these three isomers, the (1–2,3–4,5–6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin‐TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF‐like disulfide‐bonding pattern (1–3,2–4,5–6) was not found among the oxidation products.

The domain with the connecting region amino acids (DIDE) at the C‐terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide‐bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin‐binding affinities of these isomers to the isomer with the EGF‐like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF‐like pattern. Thrombin‐binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1–2,3–4,5–6). This isomer doubled the clotting time at a concentration of 200 nM and showed a Ki for protein C activation of 2 μM, both an order of magnitude better than the isomer with EGF‐like disulfide bonds.
Production, purification, and crystallization of human interleukin‐1β converting enzyme derived from an Escherichia coli expression system
Authors: Malinowski John J. , Grasberger Bruce L. , Trakshel Gary , Huston Edward E. , Banks Tracey M. , Brake Patricia G. , Ciccarelli Richard B. , Jones Barry N. , Koehn James A. , Kratz Diane , Lundberg Nicole , Stevis Panayiotis E. , Helaszek Carla T. , Ator Mark A. , Small Wood Angela M. , Stams Travis , Rubin Byron , Alexander Richard S.
Published: February 2009

Interleukin‐1β converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL‐1β to an active mature form. The mature form of IL‐1β is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273–277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter‐based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X‐ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.
Homology modeling of an immunoglobulin‐like domain in the Saccharomyces cerevisiae adhesion protein α‐agglutinin
Authors: Lipke Peter N. , Chen Min‐Hao , Nobel Hans De , Kurjan Janet , Kahn Peter C.
Published: February 2009

The Saccharomyces cerevisiae adhesion protein α‐agglutinin is expressed by cells of α mating type. On the basis of sequence similarities, α‐agglutinin has been proposed to contain variable‐type immunoglobulin‐like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence‐based alignment algorithms impossible. We have therefore developed a secondary structure‐based method that allowed homology modeling of α‐agglutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.
Alignment of 700 globin sequences: Extent of amino acid substitution and its correlation with variation in volume
Authors: Kapp Oscar H. , Moens Luc , Vanfleteren Jaak , Trotman Clive N.A. , Suzuki Tomohiko , Vinogradov Serge N.
Published: February 2009

Seven‐hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation ∼2–3%, the variation in volume per position ranges from ∼13% for the internal to ∼21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pair‐wise similarity scores. The scores for globin‐globin comparisons over the 84 common positions overlap the globin‐phycocyanin and globin‐colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three‐on‐three α‐helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.
Side‐chain conformational entropy in protein folding
Authors: Doig Andrew J. , Sternberg Michael J. E.
Published: February 2009

An important, but often neglected, contribution to the thermodynamics of protein folding is the loss of entropy that results from restricting the number of accessible side‐chain conformers in the native structure. Conformational entropy changes can be found by comparing the number of accessible rotamers in the unfolded and folded states, or by estimating fusion entropies. Comparison of several sets of results using different techniques shows that the mean conformational free energy change (TΔS) is 1 kcal‐mol−1 per side chain or 0.5 kcal‐mol−1 per bond. Changes in vibrational entropy appear to be negligible compared to the entropy change resulting from the loss of accessible rotamers. Side‐chain entropies can help rationalize α‐helix propensities, predict protein/inhibitor complex structures, and account for the distribution of side chains on the protein surface or interior.
Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus)
Authors: Itoh Susumu , Navia Manuel A.
Published: February 2009

The consequences of site‐directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of “conservative” and “nonconservative” substitutions on the X‐ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110‐fold and 180‐fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506‐mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so‐called “40s loop” that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42‐mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.
Modeling of the structure of the Haemophilus influenzae heme‐binding protein suggests a mode of heme interaction
Authors: Dunten Pete , Mowbray Sherry L.
Published: February 2009

The structure and function of the periplasmic heme‐binding protein HbpA of Haemophilus influenzae were investigated. This protein is involved in the import of heme into the bacteria through the inner membrane, and thus is a key element of the organism's ability to survive in blood. A high degree of sequence similarity between HbpA and the dipeptide‐binding protein of Escherichia coli is suggested to be the result of a functional relationship. An HbpA model built using the dipeptide‐binding protein suggests a mode of heme binding that is distinct from those known in proteins of the human host. These results provide a starting point for rational drug design.
Homology modeling and molecular dynamics simulation of human prothrombin fragment 1
Authors: Li Leping , Darden Tom , Foley Charles , Hiskey Richard , Pedersen Lee
Published: February 2009

The crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hf1/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long‐range ionic forces was essential to reach a stable solution structure. The γ‐carboxyglutamic acid (Gla) domain and the kringle domain of hf1/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K‐dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.
Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities
Authors: Toney Michael D. , Pascarella Stefano , De Biase Daniela
Published: February 2009

A homology model for the pig isozyme of the pyridoxal phosphate‐dependent enzyme γ‐aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the pro‐S proton at C4 of GABA, which abstracted in the 1,3‐azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic ω‐amino acids such as GABA. In the proposed structure for the L‐glutamate external aldimine, the α‐carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on ω‐amino acids in one half‐reaction and on α‐amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of β‐substituted phenyl and p‐chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism‐based inhibitor vinyl‐GABA, an effective anti‐epileptic drug known clinically as Vigabatrin, would orient the scissile C4‐H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4‐H bond toward Arg 445. The active site model presented provides a basis for site‐directed mutagenesis and drug design experiments.
Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure
Authors: Kreisberg Racheli , Buchner Virginia , Arad Dorit
Published: February 2009

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural‐cysteine‐mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide‐bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three‐dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.
Crystallization and preliminary X‐ray analysis of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase
Authors: Istvan Eva S. , Hasemann Charles A. , Kurumbail Ravi G. , Uyeda Kosaku , Deisenhofer Johann
Published: February 2009

Diffraction‐quality crystals of the bifunctional enzyme fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATPγS, fructose 6‐phosphate, the detergent n‐octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 Å and c = 130.6 Å. Flash‐frozen crystals diffract to beyond 2.2 Å, and native data have been collected.
N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose
Authors: Oikonomakos N. G. , Kontou M. , Zographos S. E. , Watson K. A. , Johnson L. N. , Bichard C. J. F. , Fleet G. W. J. , Acharya K. R.
Published: February 2009

Structure‐based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α‐D‐glucose (Ki = 1.7 mM). The best inhibitor in the class of N‐acyl derivatives of β‐D‐glucopyranosylamine, N‐acetyl‐β‐D‐glucopyranosylamine (1‐GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1‐GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 μM) and the α (Ki = 35 μM) forms of the enzyme with respect to glucose 1‐phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1‐GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T‐state conformation. Co‐crystals of the phosphorylase b‐1‐GlcNAc‐IMP complex were grown in space group P43212, with native‐like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1‐GlcNAc binds tightly at the catalytic site of T‐state phosphorylase b at approximately the same position as that of α‐D‐glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T‐state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T‐state phosphorylase b‐1‐GlcNAc‐IMP complex structure is overall similar to the T‐state phosphorylase b‐α‐D‐glucose complex structure. The structure of the 1‐GlcNAc complex provides a rational for the biochemical properties of the inhibitor.
Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects
Authors: Assemat Karine , Clément‐Métral Jenny , Alzari Pedro M.
Published: February 2009

The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, IIe, Tyr) by site‐directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA− background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45‐60% that of the wild type at 37°, and essentially identical at 42°. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37°, demonstrating that subtle modifications of the protein interior at position 25 (Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro.
Chemically crosslinked protein dimers: Stability and denaturation effects
Authors: Byrne Michael P. , Stites Wesley E.
Published: February 2009

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1, 6‐bismaleimidohexane (BMH), 1, 3‐dibromo‐2‐propanol (DBP), and the chemical warfare agent, mustard gas (bis(2‐chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two‐state model of unfolding. The monofunctional reagent, e‐maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three‐state thermodynamic model of two successive transitions in which the individual sub‐units cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two‐state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.
Significant discrepancies between van't hoff and calorimetric enthalpies. II
Authors: Liu Yufeng , Sturtevant Julian M.
Published: February 2009

Isothermal calorimetric titration of 18‐crown‐6 ether with BaCl2 in pure aqueous solution over the temperature range 7‐40 °C gives precise binding constants and enthalpy changes. Nonlinear least‐squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature‐independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.
The role of a conserved tyrosine residue in high‐potential iron sulfur proteins
Authors: Iwagami Sakura G. , Creagh A. Louise , Haynes Charles A. , Borsari Marco , Felli Isabella C. , Piccioli Mario , Eltis Lindsay D.
Published: February 2009

Conserved tyrosine‐12 of Ectothiorhodospira halophila high‐potential iron sulphur protein (HiPIP) iso‐I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild‐type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0‐11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr‐12 hydroxyl group with Asn‐14 Oδ1 and Lys‐59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys‐59/Val‐58 peptide bond, thereby perturbing Gly‐46. The ΔΔGappD of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 °C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr‐12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 °C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr‐12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.
Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy
Authors: Frank M. Kirsten , Clore G. Marius , Gronenborn Angela M.
Published: February 2009

The structure and dynamics of the urea‐denatured B1 immunoglobulin binding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 1H, 15N, and 13C assignments are obtained by means of sequential through‐bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHNα coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. 15N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second β‐hairpin, the N‐terminus of the α‐helix, and the middle of the α‐helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (∼3 ns) and could range from about 6 ns in the case of one model to between 4 μs and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N‐ and C‐termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5.