bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation
Authors: Neet Kenneth E. , Timm David E.
Published: February 2009

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2‐state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, δGu(H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing Gu(H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.
Six new candidate members of the α/β twisted open‐sheet family detected by sequence similarity to flavodoxin
Authors: Grandori Rita , Carey Jannette
Published: February 2009

Strong sequence similarity has been reported among WrbA (the Trp repressor‐binding protein of Escherichia coli); Ycp4, a protein of unknown function from the budding yeast Saccharomyces cerevisiae; P25, the pap1‐dependent protein of the fission yeast Schizosaccharomyces pombe; and the translation product of a partial cDNA sequence from rice seedling root (Oryza sativa, locus Ricr02421a; here referred to as RicR). Further homology search with the profile method indicates that all the above sequences are related to the flavodoxin family and, in turn, allows detection of the recently proposed flavodoxin‐like proteins from E. coli, MioC and the hypothetical protein YihB. We discuss sequence conservation with reference to the known 3‐dimensional structures of flavodoxins. Conserved sequence and hydrophobicity patterns, as well as residue‐pair interaction potentials, strongly support the hypothesis that these proteins share the α/β twisted open‐sheet fold typical of flavodoxins, with an additional α/β unit in the WrbA family. On the basis of the proposed structural homology, we discuss the details of the putative FMN‐binding sites. Our analysis also suggests that the helix‐turn‐helix motif we identified previously in the C‐terminal region of the WrbA family is unlikely to reflect a DNA‐binding function of this new protein family.
A revised set of potentials for β‐turn formation in proteins
Authors: Hutchinson E. Gail , Thornton Janet M.
Published: February 2009

Three thousand eight hundred ninety‐nine β‐turns have been identified and classified using a nonhomologous data set of 205 protein chains. These were used to derive β‐turn positional potentials for turn types I' and II' for the first time and to provide updated potentials for formation of the more common types I, II, and VIII. Many of the sequence preferences for each of the 4 positions in turns can be rationalized in terms of the formation of stabilizing hydrogen bonds, preferences for amino acids to adopt a particular conformation in ϕ, ψ space, and the involvement of turn types I' and II' in β‐hairpins. Only 1,632 (42%) of the turns occur in isolation; the remainder have at least 1 residue in common with another turn and have hence been classified as multiple turns. Several types of multiple turn have been identified and analyzed.
Three‐Dimensional structure of schistosoma japonicum glutathione s‐transferase fused with a six‐amino acid conserved neutralizing epitope of gp41 from hiv
Authors: Lim Kap , Ho Joseph X. , Keeling Kim , Gilliland Gary L. , Ji Xinhua , Rüker Florian , Carter Daniel C.
Published: February 2009

The 3‐dimensional crystal structure of glutathione S‐transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV‐1) (Muster T et al., 1993, J Virol 67:6642–6647) was determined at 2.5 Å resolution. The structure of the 3‐3 isozyme rat GST of the μ gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169–10184) was used as a molecular replacement model. The structure consists of a 4‐stranded β‐sheet and 3 α‐helices in domain 1 and 5 α‐helices in domain 2. The space group of the Sj GST crystal is P43212, with unit cell dimensions of a = b = 94.7 Å, and c = 58.1 Å. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2‐fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu‐Leu‐Asp‐Lys‐Trp‐Ala) fused to the C‐terminus of Sj GST forms a loop stabilized by symmetry‐related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes μ, α, and π. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.
Crystal structure of p‐hydroxybenzoate hydroxylase reconstituted with the modified fad present in alcohol oxidase from methylotrophic yeasts: Evidence for an arabinoflavin
Authors: Van Berkel Willem J. H. , Eppink Michel H.M. , Schreuder Herman A.
Published: February 2009

The flavin prosthetic group (FAD) of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p‐hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme–substrate complex of modified FAD‐containing p‐hydroxybenzoate hydroxylase diffract to 2.1 Å resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a‐FAD) is located in a cleft outside the active site as recently observed in several other p‐hydroxybenzoate hydroxylase complexes.

Like the native enzyme, a‐FAD‐containing p‐hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKa = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s−1). The oxidative part of the catalytic cycle of a‐FAD‐containing p‐hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4‐dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p‐hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin “out” conformation is associated with the oxidase activity.
The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin
Authors: Vijayalakshmi J. , Padmanabhan K.P. , Tulinsky A. , Mann K.G.
Published: February 2009

The X‐ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of α‐thrombin, has been determined at 2.0 Å resolution complexed with hirugen. The structure has been refined to a final R‐value of 0.169 using 14,211 observed reflections in the resolution range 8.0–2.0 Å. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen–thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186–Asp 194, Gly 216–Gly 223, Gly 142–Pro 152, and the Arg 15–Ile 16 cleavage region). The formation of the Ile 16–Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved.

With the determination of isomorphous structures of hirugen–thrombin and d‐Phe‐Pro‐Arg chloromethyl ketone (PPACK)–thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190–Gly 197 segment in the active site has an average RMS difference of 0.55 Å between the 2 structures (about 3.7σ compared to the bulk structure). This segment has 2 type II β‐bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen–thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein‐C, thrombin platelet receptor). In PPACK–thrombin, the side chain of Asp 189 and the segment Arg 221A–Gly 223 move to provide space for the inhibitor, whereas in hirugenthrombin, the Ala 190–Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.
The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules
Authors: Zegers Ingrid , Maes Dominique , Dao‐Thi Minh‐Hoa , Wyns Lode , Poortmans Freddy , Palmer Rex
Published: February 2009

The interactions of RNase A with cytidine 3′‐monophosphate (3′‐CMP) and deoxycytidyl‐3′,5′‐deoxyadenosine (d(CpA)) were analyzed by X‐ray crystallography. The 3′‐CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein–inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2′ oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3′‐CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N‐terminal helix to the rest of the protein and in the stabilization of the active site.
Residue–Residue contact substitution probabilities derived from aligned three‐dimensional structures and the identification of common folds
Authors: Rodionov Michael A. , Johnson Mark S.
Published: February 2009

We report the derivation of scores that are based on the analysis of residue‐residue contact matrices from 443 3‐dimensional structures aligned structurally as 96 families, which can be used to evaluate sequence‐structure matches. Residue–residue contacts and the more than 3 × 106 amino acid substitutions that take place between pairs of these contacts at aligned positions within each family of structures have been tabulated and segregated according to the solvent accessibility of the residues involved. Contact maps within a family of structures are shown to be highly conserved (∼75%) even when the sequence identity is approaching 10%. In a comparison involving a globin structure and the search of a sequence databank (>21,000 sequences), the contact probability scores are shown to provide a very powerful secondary screen for the top scoring sequence–structure matches, where between 69% and 84% of the unrelated matches are eliminated. The search of an aligned set of 2 globins against a sequence databank and the subsequent residue contact‐based evaluation of matches locates all 618 globin sequences before the first non‐globin match. From a single bacterial serine proteinase structure, the structural template approach coupled with residue–residue contact substitution data lead to the detection of the mammalian serine proteinase family among the top matches in the search of a sequence databank.
Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein
Authors: Regan Lynne , Rockwell Arlene , Wasserman Zelda , Degrado William
Published: February 2009

The introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexibility of a family of designed 4‐helix bundle proteins. The results of these analyses lend support to our working model of the proteins' structure and suggest that the proteins have limited main‐chain flexibility.
Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange
Authors: Ohguro Hiroshi , Palczewski Krzysztof , Walsh Kenneth A. , Johnson Richard S.
Published: February 2009

Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P‐Rho*). To study its conformational changes and regions interacting with P‐Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P‐Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P‐Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C‐terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P‐Rho*. These data allowed us to identify functional regions in the arrestin molecule.
Crystallization and preliminary x‐ray crystallographic analysis of the 38‐kda immunodominant antigen of mycobacterium tuberculosis
Authors: Choudhary Abha , Vyas Meenakshi N. , Vyas Nand K. , Chang Zengyi , Quiocho Florante A.
Published: February 2009

The 38‐kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody‐and T cell‐mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate‐binding protein (Mr = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38‐kDa protein has been crystallized in 2 forms that are suitable for high‐resolution structural analyses. One form belongs to the monoclinic space group P21 with unit cell dimensions of a = 67.42 Å, b = 113.38 Å, c = 42.68 Å, and β = 108.53°. The other is of the orthorhombic space group P21212 with a = 125.46 Å, b = 72.27 Å, and c = 73.43 Å. Both crystal forms diffract to about 2 Å resolution on a fine focus rotating anode.
High mobility group protein, hmg‐1, contains insignificant glycosyl modification
Authors: Bernice Chao Y. , Betty Yan S. , Scovell William M.
Published: February 2009

High mobility group protein‐1 (HMG‐1) is a ubiquitous, highly conserved, and abundant nuclear protein. Recent findings suggest that HMG‐1 may serve as a DNA chaperone protein and play a role in the regulation of transcription. There is a mounting interest in elucidating the mechanism by which HMG‐1 protein takes part in these activities. HMG‐1 has been reported to undergo an extensive array of posttranslational modifications, including glycosylation. We extend the earlier findings on the glycosylation of HMG‐1 by quantitating the amount of carbohydrate on HMG‐1 from calf thymus and chicken erythrocytes isolated by 2 different purification procedures. In addition, 2 different developmental stages (embryonic and adult) were examined in the chicken erythrocytes. The glycosyl composition was quantitated using the Dionex HPAE‐PAD II system. Furthermore, the presence of O‐linked GlcNAc on HMG‐1 was determined by the enzymatic incorporation of 3H‐galactose into HMG‐1 protein. Contrary to earlier reports, less than 0.5 mol of total monosaccharides (Fuc, Man, GalNH2, GlcNH2, Gal) were detected per mole of HMG‐1 protein, regardless of the source of the protein or the method of isolation. In addition, less than 0.002 mol of O‐linked GlcNAc per mole of HMG‐1 protein was detected. Thus, insignificant amount of glycosylation was found on HMG‐1 protein. Because O‐linked GlcNAc modification of proteins is believed to be a reversible posttranslational event, more definitive studies will need to be conducted before ruling out that the function of HMG‐1 protein is not regulated by glycosylation.
The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences
Authors: Grishin Nick V. , Phillips Margaret A.
Published: February 2009

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three‐dimensional structure was known for at least two members of each family. The analysis indicates that the sub‐unit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.
Interactions between hydrophobic side chains within α‐helices
Authors: Creamer Trevor P. , Rose George D.
Published: February 2009

The thermodynamic basis of helix stability in peptides and proteins is a topic of considerable interest. Accordingly, we have computed the interactions between side chains of all hydrophobic residue pairs and selected triples in a model helix, using Boltzmann‐weighted exhaustive modeling. Specifically, all possible pairs from the set Ala, Cys, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val were modeled at spacings of (i, i + 2), (i, i + 3), and (i, i + 4) in the central turn of a model poly‐alanyl α‐helix. Significant interactions —both stabilizing and destabilizing —were found to occur at spacings of (i, i + 3) and (i, i + 4), particularly in side chains with rings (i.e., Phe, Tyr, Trp, and His). In addition, modeling of leucine triples in a helix showed that the free energy can exceed the sum of pairwise interactions in certain cases. Our calculated interaction values both rationalize recent experimental data and provide previously unavailable estimates of the constituent energies and entropies of interaction.
Thermodynamics of the temperature‐induced unfolding of globular proteins
Authors: Khechinashvili Nikolay N. , Janin Joël , Rodier Francis
Published: February 2009

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature‐induced unfolding of 11 globular proteins of known three‐dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent‐accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 °C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non‐polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature‐induced unfolding.
Simple models for the analysis of binding protein‐dependent transport systems
Authors: Shilton Brian H. , Mowbray Sherry L.
Published: February 2009

Mathematical modeling was used to evaluate experimental data for bacterial binding protein‐dependent transport systems. Two simple models were considered in which ligand‐free periplasmic binding protein interacts with the membrane‐bound components of transport. In one, this interaction was viewed as a competition with the ligand‐bound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself. Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system. By contrast, a model that omitted the interaction of ligand‐free binding protein did not fit the experimental data. Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters. Practical suggestions are given for further experimental design.
Comparison of and limits of accuracy for statistical analyses of vibrational and electronic circular dichroism spectra in terms of correlations to and predictions of protein secondary structure
Authors: Pancoska Petr , Bitto Eduard , Janota Vit , Urbanova Marie , Gupta Vijai P. , Keiderling Timothy A.
Published: February 2009

This work provides a systematic comparison of vibrational CD (VCD) and electronic CD (ECD) methods for spectral prediction of secondary structure. The VCD and ECD data are simplified to a small set of spectral parameters using the principal component method of factor analysis (PC/FA). Regression fits of these parameters are made to the X‐ray‐determined fractional components (FC) of secondary structure. Predictive capability is determined by computing structures for proteins sequentially left out of the regression. All possible combinations of PC/FA spectral parameters (coefficients) were used to form a full set of restricted multiple regressions with the FC values, both independently for each spectral data set as well as for the two VCD sets and all the data grouped together. The complete search over all possible combinations of spectral parameters for different types of spectral data is a new feature of this study, and the focus on prediction is the strength of this approach. The PC/FA method was found to be stable in detail to expansion of the training set. Coupling amide II to amide I' parameters reduced the standard deviations of the VCD regression relationships, and combining VCD and ECD data led to the best fits. Prediction results had a minimum error when dependent on relatively few spectral coefficients. Such a limited dependence on spectral variation is the key finding of this work, which has ramifications for previous studies as well as suggests future directions for spectral analysis of structure. The best ECD prediction for helix and sheet uses only one parameter, the coefficient of the first subspectrum. With VCD, the best predictions sample coefficients of both the amide I' and II bands, but error is optimized using only a few coefficients. In this respect, ECD is more accurate than VCD for α‐helix, and the combined VCD (amide I'+II) predicts the β‐sheet component better than does ECD. Combining VCD and ECD data sets yields exceptionally good predictions by utilizing the strengths of each. However, the residual error, its distribution, and, most importantly, the lack of dependence of the method on many of the significant components derived from the spectra leads to the conclusion that the heterogeneity of protein structure is a fundamental limitation to the use of such spectral analysis methods. The underutilization of these data for prediction of secondary structure suggests spectral data could predict a more detailed descriptor.
The SEA module: A new extracellular domain associated with O‐glycosylation
Authors: Bork Peer , Patthy Laszlo
Published: February 2009

Using a variety of homology search methods and multiple alignments, a new extracellular module was identified in (1) agrin, (2) enterokinase, (3) a 63‐kDa sea urchin sperm protein, (4) perlecan, (5) the breast cancer marker MUC1 (episialin), (6) the cell surface antigen 114/A10, and (7/8) two functionally uncharacterized, probably extracellular, Caenorhabditis elegans proteins. Despite the functional diversity of these adhesive proteins, a common denominator seems to be their existence in heavily glycosylated environments. In addition, the better characterized proteins mentioned above contain all O‐glycosidiclinked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties.
Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability
Authors: Fairman Robert , Chao Hann‐Guang , Mueller Luciano , Lavoie Thomas B. , Shen Liyang , Novotny Jiri , Matsueda Gary R.
Published: February 2009

Limited information is available on inherent stabilities of four‐chain coiled‐coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four‐chain coiled‐coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four‐chain coiled‐coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain α‐helical chains 21, 28, and 35 amino acids in length are 3.1 × 10−12, 6.7 × 10−23, and 1.0 × 10−38 M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 μM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled‐coil formation in light of the work presented here.
Solution structure of the potassium channel inhibitor agitoxin 2: Caliper for probing channel geometry
Authors: Krezel Andrzej M. , Kasibhatla Chandrasekhar , Hidalgo Patricia , Mackinnon Roderick , Wagner Gerhard
Published: February 2009

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple‐stranded antiparallel β‐sheet and a single helix covering one face of the β‐sheet. The cysteine side chains connecting the β‐sheet and the helix form the core of the molecule. One edge of the β‐sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface‐exposed residues, Arg24, Lys27, and Arg31 of the β‐sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel‐binding sites of agitoxin 2 formed a basis for site‐directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel‐toxin interactions.
Escherichia coli alkaline phosphatase: X‐ray structural studies of a mutant enzyme (His‐412 → Asn) at one of the catalytically important zinc binding sites
Authors: Ma Lan , Tibbitts Thomas T. , Kantrowitz Evan R.
Published: February 2009

The X‐ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His‐412 was replaced by Asn has been determined at both low (‐Zn) and high (+Zn) concentrations of zinc. In the wild‐type structure, His‐412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn,) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614–31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild‐type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N(+Zn) enzyme was refined to an R‐factor of 0.174 at 2.2 Å resolution, and the structure of the H412N(‐Zn) enzyme was refined to an R‐factor of 0.166 at a resolution of 2.6 Å. Both indicated that the Asn residue substituted for His‐412 did not coordinate well to Zn1. In the H412N(‐Zn) structure, the Zn, site had very low occupancy and the phosphate was shifted by 1.8 Å from its position in the wild‐type structure. The Mg binding site was also affected by the substitution of Asn for His‐412. Both structures of the H412N enzyme also revealed a surface‐accessible cavity near the Zn, site that may serve as a binding site for Tris. The binding of Tris at the Zn, site may block Zn2+ binding at this site, which would account for the reduced affinity of the H412N enzyme for substrate as well as its reduced catalytic efficiency in the presence of Tris. Thus, the replacement of His‐412 with Asn affects all three metal binding sites, suggesting that the relative positions of the metals bound at the active site of E. coli alkaline phosphatase are strongly linked and critical for efficient catalysis.
The optimization of protein‐solvent interactions: Thermostability and the role of hydrophobic and electrostatic interactions
Authors: Spassov Velin Z. , Karshikoff Andrej D. , Ladenstein Rudolf
Published: February 2009

Protein‐solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent‐accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three‐dimensional structure available in the Protein Data Bank. The dependence of the total solvent‐accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent‐accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein‐solvent and charge‐charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge‐charge interactions.
Control of aggregation in protein refolding: A variety of surfactants promote renaturation of carbonic anhydrase II
Authors: Wetlaufer D.B. , Xie Y.
Published: February 2009

The denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of ∼0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle‐forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.
Gibbs motif sampling: Detection of bacterial outer membrane protein repeats
Authors: Neuwald Andrew F. , Liu Jun S. , Lawrence Charles E.
Published: February 2009

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif‐encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix‐turn‐helix DNA‐binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403–410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric β‐barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane‐spanning β‐strands. These β‐strands occur on the membrane interface (as opposed to the trimeric interface) of the β‐barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles.
Pseudotorsional OCCO backbone angle as a single descriptor of protein secondary structure
Authors: Laiter Sergei , Vaisman Iosif I. , Tropsha Alexander , Hoffman Doug L. , Singh Raj K.
Published: February 2009

Protein secondary structure is conventionally identified using characteristic ranges of two backbone torsional angles π and Ψ We suggest that the secondary structure can be adequately characterized by a single descriptor, the Oi‐1 Ci‐1CiOi (where i is the residue number) pseudotorsional backbone angle. A set of 102 structurally distinct protein chains from the Protein Data Bank was used to evaluate the adequacy of this descriptor. We find that a specific range of OCCO angles corresponds to each major secondary structure. The complete range of OCCO angles (‐180° to 179°) was broken into 18 consecutive subranges of 20° each, and each subrange was assigned a letter. Thus, the OCCO profiles for each protein in the database were “translated” into a sequence of letters. The Needleman‐Wunsch primary sequence alignment algorithm was then used for secondary/tertiary structure comparison and alignment. Preliminary results indicate that this new approach has a significant potential for rapid identification of fold families in the Protein Data Bank.
Crystallization and preliminary X‐ray analysis of electron transfer flavoproteins from human and paracoccus denitrificans
Authors: Roberts David L. , Kim Jung‐Ja P. , Herrick Kevin R. , Frerman Frank E.
Published: February 2009

Mammalian electron transfer flavoprotein (ETF) is a soluble, heterodimeric flavoprotein responsible for the oxidation of at least nine primary matrix flavoprotein dehydrogenases. Crystals have been obtained for the recombinant human electron transfer flavoprotein (ETFhum) by the sitting‐drop vapor diffusion technique using polyethylene glycol (PEG) 1500 at pH 7.0 as the precipitating agent. ETFhum crystallizes in the monoclinic space group P21, with unit cell parameters a = 47.46 Å, b = 104.10 Å, c = 63.79 Å, and β = 110.02°. Based on the assumption of one αβ dimer per asymmetric unit, the Vm value is 2.69 Å3/Da. A native data set has been collected to 2.1 Å resolution. One heavy‐atom derivative has also been obtained by soaking a preformed crystal of ETFhum in 2 mM thimerosal solution for 2 h at 19 °C. Patterson analysis indicates one major site. The analogous electron transfer flavoprotein from Paracoccus denitrificans (ETFpar) has also been crystallized using PEG 8000 at pH 5.5 as the precipitating agent. ETFpar crystallizes in the orthorhombic space group P212121, with unit cell parameters a = 79.98 Å, b = 182.90 Å, and c = 70.07 Å. The Vm value of 2.33 Å3/Da is consistent with two αβ dimers per asymmetric unit. A native data set has been collected to 2.5 Å resolution.
Crystal structure of thioltransferase at 2.2 Å resolution
Authors: Katti Suresh K. , Robbins Arthur H. , Yang Yanfeng , Wells William W.
Published: February 2009

We report here the first three‐dimensional structure of a mammalian thioltransferase as determined by single crystal X‐ray crystallography at 2.2 Å resolution. The protein is known for its thiol‐redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy‐atom derivatives. The protein folds into an α/β structure with a four‐stranded mixed β‐sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol‐redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22‐Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis‐peptide configuration. Mutational data available on the protein are in agreement with the three‐dimensional structure.
De novo design of the hydrophobic cores of proteins
Authors: Desjarlais John R. , Handel Tracy M.
Published: February 2009

We have developed and experimentally tested a novel computational approach for the de novo design of hydrophobic cores. A pair of computer programs has been written, the first of which creates a “custom” rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program uses a genetic algorithm to globally optimize for a low energy core sequence and structure, using the custom rotamer library as input. Success of the programs in predicting the sequences of native proteins indicates that they should be effective tools for protein design.

Using these programs, we have designed and engineered several variants of the phage 434 cro protein, containing five, seven, or eight sequence changes in the hydrophobic core. As controls, we have produced a variant consisting of a randomly generated core with six sequence changes but equal volume relative to the native core and a variant with a “minimalist” core containing predominantly leucine residues. Two of the designs, including one with eight core sequence changes, have thermal stabilities comparable to the native protein, whereas the third design and the minimalist protein are significantly destabilized. The randomly designed control is completely unfolded under equivalent conditions. These results suggest that rational de novo design of hydrophobic cores is feasible, and stress the importance of specific packing interactions for the stability of proteins. A surprising aspect of the results is that all of the variants display highly cooperative thermal denaturation curves and reasonably dispersed NMR spectra. This suggests that the non‐core residues of a protein play a significant role in determining the uniqueness of the folded structure.
Prediction of polyelectrolyte polypeptide structures using Monte Carlo conformational search methods with implicit solvation modeling
Authors: Evans John Spencer , Chan Sunney I. , Goddard William A.
Published: February 2009

Many interesting proteins possess defined sequence stretches containing negatively charged amino acids. At present, experimental methods (X‐ray crystallography, NMR) have failed to provide structural data for many of these sequence domains. We have applied the dihedral probability grid‐Monte Carlo (DPG‐MC) conformational search algorithm to a series of N‐ and C‐capped polyelectrolyte peptides, (Glu)20, (Asp)20. (PSer)20, and (PSer‐Asp)10, that represent polyanionic regions in a number of important proteins, such as parathymosin, calsequestrin, the sodium channel protein, and the acidic biomineralization proteins. The atomic charges were estimated from charge equilibration and the valence and van der Waals parameters are from DREIDING. Solvation of the carboxylate and phosphate groups was treated using sodium counterions for each charged side chain (one Na+ for COO−; two Na for CO(PO3)−2) plus a distance‐dependent (shielded) dielectric constant, ϵ = ϵ0R, to simulate solvent water. The structures of these polyelectrolyte polypeptides were obtained by the DPG‐MC conformational search with ϵ0 = 10, followed by calculation of solvation energies for the lowest energy conformers using the protein dipole‐Langevin dipole method of Warshel.

These calculations predict a correlation between amino acid sequence and global folded conformational minima:

Poly‐L‐Glu20, our structural benchmark, exhibited a preference for right‐handed α‐helix (47% helicity), which approximates experimental observations of 55–60% helicity in solution.

For Asp‐ and PSer‐containing sequences, all conformers exhibited a low preference for right‐handed α‐helix formation (≤10%), but a significant percentage (?20% or greater) of β‐strand and β‐turn dihedrals were found in all three sequence cases: (1) Aspn forms supercoil conformers, with a 2:1:1 ratio of β‐turn:β‐strand:α‐helix dihedral angles; (2) PSer20 features a nearly 1:1 ratio of β‐turn:β‐sheet dihedral preferences, with very little preference for α‐helical structure, and possesses short regions of strand and turn combinations that give rise to a collapsed bend or hairpin structure; (3) (PSer‐Asp)10 features a 3:2:1 ratio of β‐sheet:β‐turn:α‐helix and gives rise to a superturn or C‐shaped structure.
Binary patterning of polar and nonpolar amino acids in the sequences and structures of native proteins
Authors: West Michael W. , Hecht Michael H.
Published: February 2009

Protein sequences can be represented as binary patterns of polar (○) and nonpolar (•) amino acids. These binary sequence patterns are categorized into two classes: Class A patterns match the structural repeat of an idealized amphiphilic α‐helix (3.6 residues per turn), and class B patterns match the structural repeat of an idealized amphiphilic β‐strand (2 residues per turn). The difference between these two classes of sequence patterns has led to a strategy for de novo protein design based on binary patterning of polar and nonpolar amino acids. Here we ask whether similar binary patterning is incorporated in the sequences and structures of natural proteins. Analysis of the Protein Data Bank demonstrates the following. (1) Class A sequence patterns occur considerably more frequently in the sequences of natural proteins than would be expected at random, but class B patterns occur less often than expected. (2) Each pattern is found predominantly in the secondary structure expected from the binary strategy for protein design. Thus, class A patterns are found more frequently in α‐helices than in β‐strands, and class B patterns are found more frequently in β‐strands than in α‐helices. (3) Among the α‐helices of natural proteins, the most commonly used binary patterns are indeed the class A patterns. (4) Among all β‐strands in the database, the most commonly used binary patterns are not the expected class B patterns. (5) However, for solvent‐exposed β‐strands, the correlation is striking: All β‐strands in the database that contain the class B patterns are exposed to solvent. (6) The bias of class A patterns for α‐structure over β‐structure and the bias of class B patterns for β‐structure over α‐structure are significant, not merely when compared to other binary patterns of polar (○) and nonpolar (•) amino acids, but also when compared to the full range of sequences in the database. The implications for the design of novel proteins are discussed.
Posttranslational modifications of bovine osteopontin: Identification of twenty‐eight phosphorylation and three O‐glycosylation sites
Authors: Sørensen Esben S. , Petersen Torben E. , Højrup Peter
Published: February 2009

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S‐ethylcysteine‐derivatized phosphopeptides, and mass spectrometric analysis. Twenty‐five phosphoserines and one phosphothreonine were located in Ser/Thr‐X‐Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser‐X‐X‐Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11–32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O‐glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine‐and proline‐rich region of the protein. Three putative N‐glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O‐glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well‐conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.