bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: Evidence for metal‐binding cooperativity
Authors: Gehrig Peter M. , You Chunhui , Kägi Jeremias H. R. , Hunziker Peter E. , Dallinger Reinhard , Gruber Christine , Brouwer Marius 
Published: October 2010

AbstractElectrospray ionization (ESI) mass spectra of both well‐characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal‐ion‐binding modes and stoichiometries. The ESI mass spectra of the zinc‐and cadmium‐binding MTs showed a single main peak corresponding to metal‐to‐protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three‐ and four‐metal clusters. An unstable copper‐specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal‐thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal‐free human MT‐2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four‐metal cluster and of the thermodynamically less stable three‐metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal‐free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal‐free blue crab MT‐I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditits elegans MT‐II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.
Solution NMR evidence for a cis Tyr‐Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A
Authors: Xiong Ying , Swapna G.V.T. , Montelione Gaetano T. , Juminaga Darmawi , Wedemeyer William J , Scheraga Harold A. 
Published: October 2010

AbstractProline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92‐Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline‐93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92‐Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996,Biochemistry 35:1548–1559). Here, we describe the analysis of backbone proton resonance assignments for P93 A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI β‐bend conformation with a cis Tyr92‐Ala93 peptide group in the folded structure. This is in contrast to the reported X‐ray crystal structure of [Pro93Gly]‐RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998,Protein Sci 7:1620–1625), in which Tyr92‐Gly93 forms a type‐II β‐bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type‐II bend (with a positive value of ø93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type‐VI β‐bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild‐type RNase A.
Detection of intact megaDalton protein assemblies of vanillyl‐alcohol oxidase by mass spectrometry
Authors: Van Berkel Willem J. H. , Van Den Heuvel Robert H. H. , Versluis Cees , Heck Albert J. R. 
Published: October 2010

AbstractWell‐resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2%. the mass spectral data seem to reflect known solution‐phase behavior of the studied protein assembly and have therefore been directly used to probe the protein assembly topology and stability as a function of ionic strength and ph.
The prion domain of yeast Ure2P induces autocatalytic formation of amyloid fibers by a recombinant fusion protein
Authors: Schlumpberger Martin , Wille Holger , Baldwin Michael A. , Butler Darel A. , Herskowitz Ira , Prusiner Stanley B. 
Published: October 2010

AbstractThe Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion‐like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione‐S‐transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST‐UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid‐like characteristics, such as Congo red dye binding and green‐gold birefringence. The fibrils exhibited high β‐sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full‐length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self‐propagating inactive form of Ure2p. We also found that the uncleaved GST‐UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, β‐sheet‐rich conformation. The findings on the GST‐UPD fusion protein indicate that the ability of the prion domain to mediate a prion‐like conversion process is not specific for or limited to the Ure2p.
Conservation among HSP60 sequences in relation to structure, function, and evolution
Authors: Brocchieri Luciano , Karlin Samuel 
Published: October 2010

AbstractThe chaperonin HSP60 (GroEL) proteins are essential in eubacterial genomes and in eukaryotic organelles. Functional regions inferred from mutation studies and the Escherichia coli GroEL 3D crystal complexes are evaluated in a multiple alignment across 43 diverse HSP60 sequences, centering on ATP/ADP and Mg2+ binding sites, on residues interacting with substrate, on GroES contact positions, on interface regions between monomers and domains, and on residues important in allosteric conformational changes. The most evolutionary conserved residues relate to the ATP/ADP and Mg2+ binding sites. Hydrophobic residues that contribute in substrate binding are also significantly conserved. A large number of charged residues line the central cavity of the GroEL—GroES complex in the substrate‐releasing conformation. These span statistically significant intra‐ and inter‐monomer three‐dimensional (3D) charge clusters that are highly conserved among sequences and presumably play an important role interacting with the substrate. Unaligned short segments between blocks of alignment are generally exposed at the outside wall of the Anfinsen cage complex. The multiple alignment reveals regions of divergence common to specific evolutionary groups. For example, rickettsial sequences diverge in the ATP/ADP binding domain and Gram‐positive sequences diverge in the allosteric transition domain. The evolutionary information of the multiple alignment proffers attractive sites for mutational studies.
The X‐ray structure of a chitinase from the pathogenic fungus Coccidioides immitis
Authors: Hollis Thomas , Monzingo Arthur F. , Bortone Kara , Ernst Stephen , Robertus Jon D. , Cox Rebecca 
Published: October 2010

AbstractThe X‐ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 Å resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight‐stranded β/α‐barrel. Although lacking an N‐terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.
Fluorinated alcohol, the third group of cosolvents that stabilize the molten‐globule state relative to a highly denatured state of cytochrome c
Authors: Konno Takashi , Iwashita Jun , Nagayama Kuniaki 
Published: October 2010

AbstractThe effects of 1,1,1,3,3,3‐hexafluoro‐isopropanol (HFIP) on the conformation of cytochrome c (cyt c) at pH 1.9 were studied using a combination of spectroscopic and physical methods. Analysis varying the HFIP concentration showed that a compact denatured conformation (MHF) accumulates in a low concentration range of HFIP in the middle of structural transition from the highly unstructured acid‐denatured state to the highly helical alcohol‐denatured state of cyt c. This contrasts clearly with the effect of isopropanol (IP), in which no compact conformation accompanied with the transition. Analysis varying concentrations of HFIP and NaCl concurrently showed that the MHF state of cyt c is essentially identical to the salt‐induced molten‐globule (MG) state, and the MG state in the presence of salt was also stabilized by a low concentration of HFIP. Furthermore, 2,2,2‐trifluoroethanol stabilized MHF similarly to HFIP, supporting the proposition that the specific effect observed for HFIP is caused by fluorination of alcohol. The mechanism stabilizing compact conformation by HFIP remains unclear, but is probably distinct from that of salts and polyols, which are also known to stabilize the MG‐like state.
Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)—Identification of structural features important for the lethal action of snake venom cardiotoxins
Authors: Jayaraman Gurunathan , Kumar Thallampuranam Krishnaswamy Suresh , Tsai Chung‐Chu , Srisailam Sampath , Yu Chin , Chou Shan‐Ho , Ho Chewn‐Lang 
Published: October 2010

AbstractThe aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double‐stranded antiparallel β‐sheet domains. Critical examination of the threedimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct “finger‐shaped” projection comprising of nonpolar residues. The occurrence of the nonpolar “finger‐shaped” projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the “finger‐shaped” projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar “finger‐shaped” projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.
An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution
Authors: Kumaran Sangaralingam , Datta Debjani , Roy Rajendra P. 
Published: October 2010

AbstractWe report an enigmatic peptide ligation reaction catalyzed by Glu‐specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free α‐carboxyl and α‐amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 °C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size‐exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length‐dependent enhancement of secondary structure in the oligomers, suggesting that protease‐catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the “conformational trapping” of the product. The ligation reaction proceeded in two phases—slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two‐step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product‐directed “conformational trap” as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a “V8 peptide ligase.”
Determination of α‐helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory
Authors: Sun Jia Ke , Penel Simon , Doig Andrew J. 
Published: October 2010

AbstractSurveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the α‐helix. We have therefore extended helix‐coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N‐cap, C‐cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac‐XAKAAAAKAAGY‐CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol−1 compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol−1 less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.
Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion
Authors: Saridakis Emmanuel , Chayen Naomi E. 
Published: October 2010

AbstractA simple method for growing protein crystals in the metastable zone using the vapor diffusion technique is described. The coverslips holding the hanging drops are transferred, after being incubated for some time at conditions normally giving many small crystals, over reservoirs at concentrations that normally yield clear drops. Fewer, much larger and better diffracting crystals are obtained, compared with conventional crystallization at similar conditions. To our knowledge, this is the first report of a significant crystal improvement due to “backing off” from nucleation conditions, using the hanging drop method.A correlation of the transfer time with published results for vapor diffusion equilibration of poly(ethylene glycol) solutions is also presented.
Scoring functions in protein folding and design
Authors: Dima Ruxandra I. , Banavar Jayanth R. , Maritan Amos 
Published: October 2010

AbstractWe present an analysis of the assumptions behind some of the most commonly used methods for evaluating the goodness of the fit between a sequence and a structure. Our studies on a lattice model show that methods based on statistical considerations are easy to use and can capture some of the features of protein‐like sequences and their corresponding native states, but unfortunately are incapable of recognizing, with certainty, the native‐like conformation of a sequence among a set of decoys. Meanwhile, an optimization method, entailing the determination of the parameters of an effective free energy of interaction, is much more reliable in recognizing the native state of a sequence. However, the statistical method is shown to perform quite well in tests of protein design.
Novel inter‐protein cross‐link identified in the GGH‐ecotin D137Y dimer
Authors: Person Maria D. , Brown Kathlynn C. , Mahrus Sami , Craik Charles S. , Burlingame Alma L. 
Published: October 2010

AbstractIn the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl–glycyl–histidine (GGH) mediates efficient and specific oxidative protein cross‐linking. The fusion of GGH to the N terminus of a protein allows for the cross‐linking reagent to be delivered in a site‐specific fashion, making this system extremely useful for analyzing protein–protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross‐linking efficiency. Cross‐linking increased four‐fold for GGH‐ecotin D137Y in comparison to wild‐type GGH‐ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross‐linked GGH‐ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross‐link between the N‐terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross‐link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross‐linking yields, and suggests that the cross‐linking reagent may be of more general utility than previously thought.
Functionally relevant motions of haloalkane dehalogenases occur in the specificity‐modulating cap domains
Authors: Otyepka Michal , Damborský Jiří 
Published: October 2010

AbstractOne‐nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity‐modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the α4′‐helix‐loop‐α4‐helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide‐stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.
CYP1, a hypovirus‐regulated cyclophilin, is required for virulence in the chestnut blight fungus
Authors: CHEN MIN‐MEI , JIANG MINGGUO , SHANG JINJIE , LAN XIUWAN , YANG FENG , HUANG JINGKUAN , NUSS DONALD L. , CHEN BAOSHAN 
Published: October 2010

SUMMARYCyclophilins are peptidyl‐prolyl cis–trans isomerases that are highly conserved throughout eukaryotes and are the cellular target of the immunosuppressive drug cyclosporin A (CsA). We cloned cyp1, a cyclophilin A‐encoding gene in the phytopathogenic fungus Cryphonectria parasitica, and showed that this gene was downregulated following infection by a virulence‐attenuating hypovirus. The function of cyp1 was further investigated by construction of a cyp1 deletion mutant. Although the wild‐type C. parasitica strain EP155 was sensitive to CsA, the Δcyp1 strain was highly tolerant to CsA, indicating that CYP1 was the target of CsA. Deletion of cyp1 resulted in reduced virulence when inoculated to chestnut stems. Transcriptional analysis revealed that deletion of cyp1 also reduced transcript levels for genes encoding key components of the heterotrimeric guanosine triphosphate‐binding protein signalling pathway that are essential for sensing environmental cues and are involved in C. parasitica development and virulence.