bluuebunny/crossref_metadata_2025_split 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

AbstractUsing 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

AbstractUnliganded 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

AbstractThe 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

AbstractHuman 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

AbstractThe 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

AbstractThe 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

AbstractTetranectin, 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

AbstractγB‐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

AbstractThe 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

AbstractS100B 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

AbstractCooperative 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

AbstractTheenvgene 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

AbstractInterleukin‐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

AbstractCrystals 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.