bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of a leucine zipper coiled coil stabilized 1.4 kcal mol−1 by phosphorylation of a serine in the e position
Authors: Szilák László , Moitra Jaideep , Vinson Charles
Published: February 2009

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (δδGP = ‐1.4 kcal mol−1 dimer−1 or ‐0.7 kcal mol−1 residue−1). Mutagenesis studies indicate that three arginines form a network of inter‐helical (i, i' + 5; i, i' + 2) and intra‐helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (δδGP = ‐0.5 kcal mol−1 dimer−1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15‐fold higher affinity. Using a transient transfection assay, we document a PKA dependent four‐fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by δδGP = + 1.2 kcal mol−1 dimer−1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.
The co‐crystal structure of unliganded bovine α‐thrombin and prethrombin‐2: Movement of the Tyr‐Pro‐Pro‐Trp segment and active site residues upon ligand binding
Authors: Malkowski Michael G. , Martin Philip D. , Guzik Jason C. , Edwards BRIAN F.P.
Published: February 2009

Unliganded bovine α‐thrombin and prethrombin‐2 have been co‐crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo‐enzyme form of bovine α‐thrombin shows dramatic shifts in placement for the Tyr‐Pro‐Pro‐Trp segment, for Glu‐192, and for the catalytic residues His‐57 and Ser‐195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7‐19 of fibrinogen Aa with a non‐cleavable scissile bond), (2) enzyme‐inhibitor complex (D‐Phe‐Pro‐Arg chloromethylketone), (3) enzyme product complex (residues 7‐16 of fibrinopeptide A), and (4) the exosite complex (residues 53‐64 of hirudin). The structures of bovine and human prethrombin‐2 are generally similar to one another (RMS deviation of 0.68 8,) but differ significantly in the Arg‐15/Ile‐16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin‐2, analogous to that seen for trypsinogen.
Absence of a stable intermediate on the folding pathway of protein A
Authors: Bai Yawen , Karimi Afshin , Dyson H. Jane , Wright Peter E.
Published: February 2009

The B‐domain of protein A has one of the simplest protein topologies, a three‐helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C‐terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped‐flow circular dichroism and NMR hydrogen‐deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench‐flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site‐directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the I16W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The I16W mutant refolds completely within the dead time of a stopped‐flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two‐state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.
Effect of limited proteolysis on the stability and enzymatic activity of human placental S‐adenosylhomocysteine hydrolase
Authors: Huang He , Yuan Chong‐Sheng , Borchardt Ronald T.
Published: February 2009

Human placental S‐adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94—Ala95, Tyr100‐Ala101, Glu243‐Ile244, Met367‐Ala368, Gln369‐Ile370, and Gly382‐Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface‐exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, Δ(ΔGu) [ΔGu (native)—ΔGu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface‐exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X‐ray crystallographic studies currently ongoing in our laboratories.
Temperature control for kinetic refolding of heat‐denatured ovalbumin
Authors: Tani Fumito , Shirai Nobuaki , Venelle Franck , Yasumoto Kyoden , Onishi Toru , Doi Etsushiro
Published: February 2009

The folding of heat‐denatured ovalbumin, a non‐inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat‐denatured at 80°C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat‐denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat‐denatured molecules assumed the metastable non‐native conformations that were susceptible to trypsin. The non‐native species were marginally stable for several days at a low temperature, but the molecules were transformed slowly into the native conformation. Considering data from size‐exclusion chromatography and from analyses of CD, intrinsic tryptophan fluorescence, and adsorption of the dye 1‐anilinonaphthalene‐8‐sulfonate, we postulated that the non‐native species that accumulated upon rapid cooling were compact but structureless globules with disordered side chains collectively as a folding intermediate. Temperature‐jumped CD experiments revealed biphasic kinetics for the refolding process of heat‐denatured ovalbumin, with the features of increasing and subsequently decreasing amplitude of the rapid and the slow phases, respectively, with the decrease in folding temperature. The temperature dependence of the refolding kinetics indicated that the yield of renaturation was maximal at about 55°C. These findings suggested the kinetic partitioning of heat‐denatured ovalbumin between alternative fates, slow renaturation to the native state and rapid collapse to the metastable intermediate state. Analysis of disulfide pairing revealed the formation of a scrambled form with non‐native disulfide interactions in both the heat‐denatured state and the intermediate state that accumulated upon rapid cooling, suggesting that non‐native disulfide pairing is responsible for the kinetic barriers that retard the correct folding of ovalbumin.
Binding of monoclonal antibody 4B1 to homologs of the lactose permease ofEscherichia coli
Authors: Sun Jianzhong , Frillingos Stathis , Kaback H. Ronald
Published: February 2009

The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+trans‐location in the lactose permease ofEscherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996,Biochemistry35:990‐998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+symporters, reveals interesting variations. 4B1 binds to theCitrobacter freundiilactose permease orE. coliraffinose permease with resultant inhibition of transport activity. BecauseE. coliraffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position. Consistently,E. colilactose permease with Pro in place of Gly254 also binds 4B1. In contrast, 4B1 binding is not observed with eitherKlebsiella pneumoniaelactose permease orE. colisucrose permease. When the epitope is transferred fromE. colilactose permease (residues 245‐259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport. The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+translocation.
Tetranectin, a trimeric plasminogen‐binding C‐type lectin
Authors: Holtet Thor Las , Graversen Jonas Heilskov , Thøgersen Hans Christian , Etzerodt Michael , Clemmensen Inge
Published: February 2009

Tetranectin, a plasminogen‐binding protein belonging to the family of C‐type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin—as well as natural tetranectin from human plasma—was shown by chemical cross‐linking analysis and SDS‐PAGE to be a homo‐trimer in solution as are other known members of the collectin family of C‐type lectins. Biochemical evidence is presented showing that an N‐terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.
Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens
Authors: Jaenicke Rainer , Slingsby Christine , Palme Stefan
Published: February 2009

γB‐crystallin is a monomeric member of the βγ‐superfamily of vertebrate eye lens proteins. It consists of two similar domains with all‐β Greek key topology associating about an approximate two‐fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one‐ or two‐domain proteins showed that the N‐terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C‐terminal partner.

It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenyl‐alanine at position 56 of the N‐terminal domain. In order to test this hypothesis, F56 was substituted by site‐directed mutagenesis in both complete γB‐crystallin and its isolated N‐terminal domain. All mutations destabilize the N‐terminal domain to about the same extent but affect the C‐terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C‐terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for γB wild type.

These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of γB‐crystallin.
Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates
Authors: Zhang Jian‐Xin , Goldenberg David P.
Published: February 2009

The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide‐bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide‐bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild‐type and mutant proteins.
Oligomerization state of S100B at nanomolar concentration determined by large‐zone analytical gel filtration chromatography
Authors: Drohat Alexander C. , Weber David J. , Nenortas Elizabeth , Beckett Dorothy
Published: February 2009

S100B is a Ca2+‐binding protein known to be a non‐covalently associated dimer, S100B(ββ), at high concentrations (0.2‐3.0 mM) under reducing conditions. The solution structure of apo‐S100B(ββ) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C‐terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo‐S100B(ββ) as determined by NMR spectroscopy. Biochemistry 35:11577‐11588). The C‐terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric S100B(ββ). Therefore, we investigated the oligomerization state of S100B at low concentrations (1‐10,000 nM) using large‐zone analytical gel filtration chromatography with 35S‐labeled S100B. We found that S100B exists (>99%) as a non‐covalently associated dimer, S100B(ββ), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as dimeric S100B(ββ) in the presence or absence of Ca2+, and that the non‐covalent dimer is most likely the form of S100B presented to target proteins.
Identification of cooperative folding units in a set of native proteins
Authors: Wallqvist A. , Smythers G.W. , Covell D.G.
Published: February 2009

Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non‐native, i.e., non‐interacting, segments. We use a novel knowledge‐based scoring function, derived from a set of non‐homologous proteins in the Protein Data Bank, to describe the interactions among residues. This procedure is used for the structural identification of cooperative folding cores for four globular proteins: bovine pancreatic trypsin inhibitor, horse heart cytochrome c, French bean plastocyanin, and staphylococcal nuclease. The theoretical folding units are shown to correspond to regions that exhibit enhanced stability against denaturation as determined from experimental hydrogen exchange protection factors. Using a sequence similarity score for related sequences, we show that, in addition to residues necessary for enzymatic function, those amino acids comprising structurally important folding cores are also preferentially conserved during evolution. This implies that the identified folding cores may be part of an array of fundamental structural folding units.
The extracellular domain of immunodeficiency virus gp41 protein: Expression inEscherichia coli, purification, and crystallization
Authors: Wingfield Paul T. , Stahl Stephen J. , Kaufman Joshua , Zlotnick Adam , Hyde C. Craig , Gronenborn Angela M. , Clore G. Marius
Published: February 2009

Theenvgene of SIV and HIV‐1 encodes a single glycoprotein gp160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N‐terminal fusion peptide, of gp41 from HIV‐1 (residues 27‐154) and SIV (residues 27‐149) have been expressed inEscherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high a‐helical content, consistent with the predicted coil‐coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X‐ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.
Multiple conformations of a human interleukin‐3 variant
Authors: Feng Yiqing , Hood William F. , Forgey Robert W. , Abegg Ann L. , Caparon Marie H. , Thiele Barrett R. , Leimgruber Richard M. , Mcwherter Charles A.
Published: February 2009

Interleukin‐3 (IL‐3) is a cytokine that stimulates the proliferation and differentiation of hematopoietic cells. The hyperactive hIL‐3 variant SC‐55494 was shown to have at least two major conformations by high‐resolution NMR spectroscopy. Mutants of SC‐55494 were constructed in which alanine was substituted for proline in order to test the hypothesis that proline cis‐trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of prolyl peptide bond configuration on biological activity. NMR spectra of four single proline‐to‐alanine mutants (P30A, P31A, P33A, and P37A) retain doubled resonances, while spectra of the double mutant P30A/P31A and the quadruple mutant P30A/P31A/P33A/ P37A are substantially free of heterogeneity. These observations suggest that the two major conformations in SC‐55494 correspond to cis and trans isomers of either or both of the R29‐P30 and P30‐P31 peptide bonds. All six mutants had somewhat lower cell proliferative activity than SC‐55494, with relative activities ranging from 40 to 80%. The P37A mutant has a binding affinity to the low‐affinity IL‐3 receptor α‐subunit statistically equivalent to SC‐55494, while P30A, P31A, and P33A each had about two‐fold decreases, and P30A/P31A and P30A/P31A/P33A/P37A had four‐fold decreases. These findings suggest an important role for the cis configuration of either or both of the R29‐P30 and P30‐P31 peptide bonds in IL‐3 for optimal interaction with the receptor α‐subunit.
Use of a fusion protein to obtain crystals suitable for X‐ray analysis: Crystallization of a GST‐fused protein containing the DNA‐binding domain of DNA replication‐related element‐binding factor, DREF
Authors: Kuge Munekazu , Fujii Yoshifumi , Shimizu Toshiyuki , Hakoshima Toshio , Hirose Fumiko , Matsukage Akio
Published: February 2009

Crystals of glutathione‐S‐transferase (GST)‐fused protein containing the DNA‐binding domain of DNA replication‐related element‐binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X‐ray crystallographic analysis revealed that crystals of the GST‐fused protein belong to space group P6122 or P6522 with unit cell dimensions a = b = 140.4 Å, c = 93.5 Å and y = 120°, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 Å resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA‐binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well‐ordered large crystals were unsuccessful. A possible application of GST‐fusion proteins for small target proteins or domains to obtain crystals suitable for X‐ray structure determination is proposed.
Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of alzheimer's amyloid β‐protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): Engineering of inhibitors with altered specificities
Authors: Scheidig Axel J. , Hynes Thomas R. , Pelletier Laura A. , Wells James A. , Kossiakoff Anthony A.
Published: February 2009

The crystal structures of the inhibitor domain of Alzheimer's amyloid β‐protein precursor (APPI) complexed to bovine chymotrypsin (C‐APPI) and trypsin (T‐APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C‐BPTI) have been solved and analyzed at 2.1 Å, 1.8 Å, and 2.6 Å resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity‐determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity‐matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin‐APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitor variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H‐bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the “allowed” residues determined from a phage display mutagenesis selection experiment.
Backbone makes a significant contribution to the electrostatics of α/β‐barrel proteins
Authors: Raychaudhuri Soumya , Younas Fayyaz , Karplus P. Andrew , Faerman Carlos H. , Ripoll Daniel R.
Published: February 2009

The electrostatic properties of seven α/β‐barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose‐1,6‐bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the α/β‐barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of α/β‐barrel enzymes for negatively charged substrates.
Solution structure of drosomycin, the first inducible antifungal protein from insects
Authors: Landon Céline , Sodano Patrick , Hetru Charles , Hoffmann Jules , Ptak Marius
Published: February 2009

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three‐dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an α‐helix and a twisted three‐stranded β‐sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized αβ (CSαβ) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short‐ and long‐chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.
Predicting protein secondary structure with probabilistic schemata of evolutionarily derived information
Authors: Thompson Michael J. , Goldstein Richard A.
Published: February 2009

We demonstrate the applicability of our previously developed Bayesian probabilistic approach for predicting residue solvent accessibility to the problem of predicting secondary structure. Using only single‐sequence data, this method achieves a three‐state accuracy of 67% over a database of 473 non‐homologous proteins. This approach is more amenable to inspection and less likely to overlearn specifics of a dataset than “black box” methods such as neural networks. It is also conceptually simpler and less computationally costly.

We also introduce a novel method for representing and incorporating multiple‐sequence alignment information within the prediction algorithm, achieving 72% accuracy over a dataset of 304 non‐homologous proteins. This is accomplished by creating a statistical model of the evolutionarily derived correlations between patterns of amino acid substitution and local protein structure. This model consists of parameter vectors, termed “substitution schemata,” which probabilistically encode the structure‐based heterogeneity in the distributions of amino acid substitutions found in alignments of homologous proteins. The model is optimized for structure prediction by maximizing the mutual information between the set of schemata and the database of secondary structures.

Unlike “expert heuristic” methods, this approach has been demonstrated to work well over large datasets. Unlike the opaque neural network algorithms, this approach is physicochemically intelligible. Moreover, the model optimization procedure, the formalism for predicting one‐dimensional structural features, and our previously developed method for tertiary structure recognition all share a common Bayesian probabilistic basis. This consistency starkly contrasts with the hybrid and ad hoc nature of methods that have dominated this field in recent years.
Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor
Authors: Pan Hong , Barany George , Woodward Clare
Published: February 2009

Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14‐38]Abu at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by α‐amino‐n‐butyric acid (Abu). [14‐38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D°—the diffusion coefficient extrapolated to infinite dilution—is determined. From D°, a value of the hydrodynamic radius, Rh, is computed from the Stokes‐Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR‐detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non‐random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974‐13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non‐native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14‐38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14‐38]Abu relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under “molten globule” conditions.
Structural studies on membrane‐embedded influenza hemagglutinin and its fragments
Authors: Gray Cameron , Tamm Lukas K.
Published: February 2009

The mechanism of influenza virus hemagglutinin (HA)‐mediated membrane fusion has been inferred in part from studies examining pH‐induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C‐ and N‐terminal residues of the HA2 chain, respectively). To reconcile structure‐based mechanisms of HA‐mediated membrane fusion with structural implications of functional studies performed on membrane‐embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR‐FTIR) spectroscopic analyses of membrane‐embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THAF−2, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA, subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the tilting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane‐inserted fusion peptide with the crystallographically determined coiled coil probably adopt an a‐helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane‐bound HA. THA2 mediates fusion, but less efficiently and with less pH‐selectivity than HA.
Ligand binding to proteins: The binding landscape model
Authors: Miller David W. , Dill Ken A.
Published: February 2009

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand‐induced denaturation, ANS and heme‐like binding, and “lock‐and‐key” and “induced‐fit” specific binding behaviors characterized by Michaelis‐Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis‐Menten isotherms. Even low‐population binding to denatured states can cause changes in global stability, hydrogen‐exchange rates, and thermal B‐factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass‐action models.
Oligomerization properties of GCN4 leucine zipper e and g position mutants
Authors: Zeng Xiangang , Zhu Hai , Lashel Hilal A. , Hu James C.
Published: February 2009

Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g‐e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of >65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing λ repressor‐leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher‐order (>dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher‐order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g‐e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.
The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket
Authors: Sørensen Steen Bech , Breddam Klaus
Published: February 2009

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site‐directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA‐Ala‐Xaa‐OH (Xaa = Leu, Glu, Lys, or Arg) and FA‐Phe‐Xaa‐OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two‐ to three‐fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D+L272D was found to have a preference for substrates with C‐terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C‐terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C‐terminal amino acid sequence determinations.
Urea‐induced conformational changes in cold‐ and heat‐denatured states of a protein, Streptomyces subtilisin inhibitor
Authors: Konno Takashi , Kamatari Yuji O. , Kataoka Mikio , Akasaka Kazuyuki
Published: February 2009

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold‐denatured (D') and heat‐denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small‐angle X‐ray scattering (SAXS) and 1H‐NMR spectroscopy. Our results indicate that the urea‐induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3°C) occur at least in two steps. In the urea concentration range of 0‐2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2‐4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.
1H, 13C, and 15N backbone assignment and secondary structure of the receptor‐binding domain of vascular endothelial growth factor
Authors: Fairbrother Wayne J. , Champe Mark A. , Christinger Hans W. , Keyt Bruce A. , Starovasnik Melissa A.
Published: February 2009

Nearly complete sequence‐specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor‐binding domain of vascular endothelial growth factor (VEGF), a 23‐kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple‐resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA(CO)NH] and a 3D 15N‐TOCSY‐HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45°C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1Hα, 13Cα, 13Cβ, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N‐NOESY‐HSQC spectrum. Two helices and a significant amount of β‐sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192‐7197].
Cloning, expression, and crystallization of a hyperthermophilic protein that is homologous to the eukaryotic translation initiation factor, eIF5A
Authors: Kim Kyeong Kyu , Yokota Hisao , Kim Rosalind , Kim Sung‐Hou
Published: February 2009

A gene coding for a protein homologous to a translation initiation factor of eukaryotes, eIF5A, was cloned from Methanococcus jannaschii, a hyperthermophile with an optimum growth temperature of 85 °C. The protein was overexpressed, purified and crystallized. The crystals were obtained by vapor diffusion method with 8% PEG 4000 as precipitant and belong to space group P4122 with unit cell dimensions a = b = 45.52 Å and c = 155.59 Å. These crystals diffract to at least 2.2 Å resolution.
Structure of a pancreatic α‐amylase bound to a substrate analogue at 2.03 Å resolution
Authors: Qian Minxie , Spinelli Silvia , Payan FrançOise , Driguez Hugues
Published: February 2009

The structure of pig pancreatic α‐amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X‐ray structure analysis of a crystal of pig pancreatic α‐amylase (PPA, EC 3.2.1.1.) soaked with an enzyme‐resistant substrate analogue, methyl 4,4′‐dithio‐α‐maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the “second binding site.” The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite ‐1 (using a nomenclature where the site of hydrolysis is taken to be between subsites ‐1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the “flexible loop” that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 Å resolution, to an R‐factor of 16.0 (Rfree 18.5).
Oxidation of apolipoprotein(a) inhibits kringle‐associated lysine binding: The loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site
Authors: Hermann Anita , Laws William R. , Harpel Peter C.
Published: February 2009

Lipoprotein(a) [Lp(a)] is a low‐density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide‐linked to apolipoprotein B‐100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2′‐azobis‐(amidinopropane) HCI (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r‐apo(a), to bind through their LBS domains. AAPH treatment caused a time‐dependent decrease in the number of functional Lp(a) or r‐apo(a) molecules capable of binding to fibrin or lysine‐Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r‐apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle‐containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r‐apo(a) was observed as assessed by native or SDS‐PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl‐containing compounds increase the LBS affinity of Lp(a) for fibrin.
Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain
Authors: Stenlund Peter , Tibell Lena A. E. , Andersson Dick
Published: February 2009

Human extracellular superoxide dismutase (hEC‐SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC‐SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X‐ray structural determination have failed, the three‐dimensional structure of hEC‐SOD is unknown. This means that alternative strategies for structural studies are needed. The N‐terminal domain of EC‐SOD has already been studied using the fusion protein FusNN, comprised of the 49 N‐terminal amino acids from hEC‐SOD fused to human carbonic anhydrase (HCAII). The N‐terminal domain in this fusion protein forms a well‐defined three‐dimensional structure, which probably contains α‐helical elements and is responsible for the tetramerization of the protein.

In this work, we have extended the studies, using site‐directed mutagenesis in combination with size‐exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a‐helix (formed by residues 14‐32) in the N‐terminal domain is essential for the subunit interaction.