bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assembly and crystallization of the complex between the human T cell coreceptor CD8α homodimer and HLA‐A2
Authors: Gao George F. , Gerth Ulrich C. , Wyer Jessica R. , Willcox Benjamin E. , O'callaghan Christopher A. , Bell John I. , Jakobsen Bent K. , Zhang Zhihong , Jones E. Yvonne 
Published: February 2009

AbstractA strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8α was devised using codon usage alterations in the 5′ region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8α, comprising residues 1‐120 of the mature protein, excluding the signal peptide and the membrane‐proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA‐A2 heavy chain, β2‐microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV‐1 were also expressed in E. coli, refolded and purified. CD8α/HLA‐A2 complexes were formed in solution and by co‐crystallization with a stoichiometry of one CD8αα dimer to one HLA‐A2‐peptide unit.
Homologues of 26S proteasome subunits are regulators of transcription and translation
Authors: Aravind L. , Ponting Chris P. 
Published: February 2009

AbstractSingle copies of an α‐helical‐rich motif are demonstrated to be present within subunits of the large multiprotein 26S proteasome and eukaryotic initiation factor‐3 (eIF3) complexes, and within proteins involved in transcriptional regulation. In addition, p40 and p47 subunits of eIF3 are shown to be homologues of the proteasome subunit Mov34, and transcriptional regulators JAB1/pad1. Finally, the proteasome subunit S5a and the p44 subunit of the basal transcription factor IIH (TFIIH) are identified as homologues. The presence of homologous, and sometimes identical, proteins in contrasting functional contexts suggests that the large multisubunit complexes of the 26S proteasome, eIF3 and TFIIH perform overlapping cellular roles.
Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A
Authors: Pearson Matthew A. , Karplus P. Andrew , Dodge Robert W. , Laity John H. , Scheraga Harold A. 
Published: February 2009

AbstractThe Tyr92‐Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide‐intact wild‐type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92‐Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95] A variant, which is an analog of the major rate‐determining three‐disulfide intermediate in the oxidative folding of RNase A, missing the 40‐95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.
Crystallization and preliminary X‐ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H‐D‐Phe‐Phe‐Arg‐chloromethylketone
Authors: Banbula Agnieszka , Potempa Jan , Travis James , Bode Wolfram , Medrano Francisco‐Javier 
Published: February 2009

AbstractGingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X‐ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P212121) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 Å resolution, respectively. Cell dimensions are a = 51.9 Å, b = 79.9 Å, and c = 99.6 Å (P212121), and a = b = 176.6 Å, and c = 143.4 Å (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self‐rotation function, could accommodate a tetramer in the asymmetric unit.
Baseline length and automated fitting of denaturation data
Authors: Allen Devon L. , Pielak Gary J. 
Published: February 2009

AbstractTo understand relationships between protein sequence and stability, we often compare data from proteins that differ by the substitution of one amino acid. Frequently, an amino acid change causes the cooperative denaturation transitions to shift to lower temperatures, diminishing the signal from the native state. Here we show that apparent stability changes, i.e., the free energy of denaturation, ΔGD, can also be caused by a deficiency of points in the low temperature end of the transition. In addition, we suggest a method for overcoming this problem.
Structure of malonic acid‐based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data
Authors: Brandstetter Hans , Engh Richard A. , Roedern Erich Graf Von , Moroder Luis , Huber Robert , Bode Wolfram , Grams Frank 
Published: February 2009

AbstractMatrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid‐based inhibitors using the X‐ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate‐like binding mode. The proposed β‐type turn‐like conformation for the improved inhibitors was confirmed by X‐ray crystallography. The observation of nonsubstrate‐like binding confirms the original strategy for structure‐based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate‐like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500‐fold improved inhibition based on these structures.
Crystal structure of the cyclophilin‐like domain from the parasitic nematode Brugia malayi
Authors: Mikol Vincent , Ma Dong , Carlow Clotilde K.S. 
Published: February 2009

AbstractCyclophilins are a family of proteins that exhibit peptidyl‐prolyl cis‐trans isomerase activity and bind the immuno‐suppressive agent Cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin‐like domain was identified at the N‐terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R‐factor of 16.9% at 2.15 Å resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.
Thermodynamic characterization of an intermediate state of human growth hormone
Authors: Gomez‐Orellana Isbuel , Varinano Bruce , Miura‐Fraboni Judy , Milstein Sam , Paton Duncan R. 
Published: February 2009

AbstractThe thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two‐state transition was observed between 0 and 100°C. The magnitudes of the ΔH and ΔCP of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n‐propanol, 2‐propanol, or 1,2‐propanediol (propylene glycol), which suggests that the self‐association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7‐8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70‐80% propylene glycol.
Conformational change in the activation of lipase: An analysis in terms of low‐frequency normal modes
Authors: Jääskeläinen Sanna , Verma Chandra S. , Hubbard Roderick E. , Lestko Pekka , Caves Leo S.D. 
Published: February 2009

AbstractThe interfacial activation of Rhizomucor miehei lipase (RmL) involves the motion of an α‐helical region (residues 82‐96) which acts as a “lid” over the active site of the enzyme, undergoing a displacement from a “closed” to an “open” conformation upon binding of substrate. Normal mode analyses performed in both low and high dielectric media reveal that low‐frequency vibrational modes contribute significantly to the conformational transition between the closed and open conformations. In these modes, the lid displacement is coupled to local motions of active site loops as well as global breathing motions. Atomic fluctuations of the first hinge of the lid (residues 83‐84) are substantially larger in the low dielectric medium than in the high dielectric medium. Our results also suggest that electrostatic interactions of Arg86 play an important role in terms of both the intrinsic stability of the lid and its displacement, through enhancement of hinge mobility in a high dielectric medium. Additional calculations demonstrate that the observed patterns of atomic fluctuations are an intrinsic feature of the protein structure and not dependent on the nature of specific energy minima.
Excluded volume in the configurational distribution of a strongly‐denatured protein
Authors: Peterscu Andrei‐Jose , Receveur Veronique , Calmettes Patrick , Durand Dominique , Smith Jeremy C. 
Published: February 2009

AbstractThe configurational distribution of phosphoglycerate kinase (PGK) strongly‐denatured in 4M guanidine hydrochloride solution is investigated using small‐angle neutron scattering (SANS) and Monte Carlo computer simulation. It is shown that the experimental scattering profile can be represented by a random flexible chain of spheres of excess scattering density with excluded volume interactions, the best agreement being achieved when partial sphere intersection is allowed. The radius of gyration of the chain increases by a factor of 4 on denaturation, whereas the average length of segments ˜5 residues long increases by only ˜10%, consistent with a picture in which the large expansion on denaturation originates primarily from increased long‐range flexibility of the polypeptide chain. The results provide a description of the chain statistics from which the construction of starting points for simulation studies of folding of the protein can be envisaged.
From coiled coils to small globular proteins: Design of a native‐like three‐helix bundle
Authors: Bryson James W. , Desjarlais John R. , Handel Tracy M. , Degrado William F. 
Published: February 2009

AbstractA monomolecular native‐like three‐helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three‐helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine‐induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native‐like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.
Design, total synthesis, and functional overexpression of the Candida rugosa lipl gene coding for a major industrial lipase
Authors: Brocca Stefania , Schmidt‐Dannert Claudia , Schmid Rolf D. , Lotti Marian , Alberghina Lilia 
Published: February 2009

AbstractThe dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip1 gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site‐directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pasto is cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lip1 of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.
Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins
Authors: Whitelegge Julian P. , Gundersen Cameron B. , Faull Kym F. 
Published: February 2009

AbstractMembrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray‐ionization mass spectrometry (ESI‐MS) of intact intrinsic membrane proteins purified using reverse‐phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the Dl and D2 reaction‐center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI‐MS study of a G‐protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post‐translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI‐MS experiments.
Guanidine hydrochloride unfolding of a transmembrane β‐strand in FepA using site‐directed spin labeling
Authors: Klug Candice S. , Feix Jimmy B. 
Published: February 2009

AbstractWe have used the electron spin resonance (ESR) site‐directed spin‐labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn‐HCl) induced denaturation of several sites along a transmembrane β‐strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the β‐strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027‐13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a β‐barrel motif comprised of a series of antiparallel β‐strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria. We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the β‐barrel domain (Klug CS et al., 1995, Biochemistry 34:14230‐14236). The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein. This work shows that sites along the β‐strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the β‐strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn‐HCl concentrations. We conclude that, in a transmembrane β‐strand, the local environment of a given residue plays a significant role in the loss of structure at each site.
Structure and distribution of pentapeptide repeats in bacteria
Authors: Bateman Alex , Murzin Alexey G. , Teichmann Sarah A. 
Published: February 2009

AbstractWe report the discovery of a novel family of proteins, each member contains tandem pentapeptide (five residue) repeats, described by the motif A(D/N)LXX. Members of this family are both membrane bound and cytoplasmic. The function of these repeats is uncertain, but they may have a targeting or structural function rather than enzymatic activity. This family is most common in cyanobacteria, suggesting a function related to cyanobacterial‐specific metabolism. Although no experimental information is available for the structure of this family, it is predicted that the tandem pentapeptide repeats will form a right‐handed β‐helical structure. A structural model of the pentapeptide repeats is presented.
Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off‐pathway aggregation
Authors: Betts Scott D. , King Jonathan 
Published: February 2009

AbstractOff‐pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self‐association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off‐pathway aggregation of the parallel β‐coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 °C prior to warming to 20 °C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature‐shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5517‐523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off‐pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450‐455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide‐bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.
MgATP binding to the nucleotide‐binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate‐binding domains
Authors: Szpikowska Barbara K. , Sherman Mark A. , Mas Maria T. , Swiderek Kristine M. 
Published: February 2009

AbstractThe eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide‐induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of ∼ 30 kDa. Using mass spectrometry (MS) and N‐terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group I1 chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP‐PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group I1 chaperonins may share similar, functionally‐important, conformational changes. Additional conformational changes are likely to involve a flexible helix‐loop‐helix motif, which is characteristic for all group II chaperonins.