bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors limiting the performance of prediction‐based fold recognition methods
Authors: Cruz Xavier De La , Thornton Janet M.
Published: October 2010

In the past few years, a new generation of fold recognition methods has been developed, in which the classical sequence information is combined with information obtained from secondary structure and, sometimes, accessibility predictions. The results are promising, indicating that this approach may compete with potential‐based methods (Rost B et al., 1997, J Mol Biol 270:471–480). Here we present a systematic study of the different factors contributing to the performance of these methods, in particular when applied to the problem of fold recognition of remote homologues.

Our results indicate that secondary structure and accessibility prediction methods have reached an accuracy level where they are not the major factor limiting the accuracy of fold recognition. The pattern degeneracy problem is confirmed as the major source of error of these methods. On the basis of these results, we study three different options to overcome these limitations: normalization schemes, mapping of the coil state into the different zones of the Ramachandran plot, and post‐threading graphical analysis.
The use of dipolar couplings for determining the solution structure of rat apo‐S100B(ββ)
Authors: Drohat Alexander C. , Tjandra Nico , Baldisseri Donna M. , Weber David J.
Published: October 2010

The relative orientations of adjacent structural elements without many well‐defined NOE contacts between them are typically poorly defined in NMR structures. For apo‐S100B(ββ) and the structurally homologous protein calcyclin, the solution structures determined by conventional NMR exhibited considerable differences and made it impossible to draw unambiguous conclusions regarding the Ca2+‐induced conformational change required for target protein binding. The structure of rat apo‐S100B(ββ) was recalculated using a large number of constraints derived from dipolar couplings that were measured in a dilute liquid crystalline phase. The dipolar couplings orient bond vectors relative to a single‐axis system, and thereby remove much of the uncertainty in NOE‐based structures. The structure of apo‐S100B(ββ) indicates a minimal change in the first, pseudo‐EF‐hand Ca2+ binding site, but a large reorientation of helix 3 in the second, classical EF‐hand upon Ca2+ binding.
Structural and phylogenetic characterization of human SLURP‐1, the first secreted mammalian member of the Ly‐6/uPAR protein superfamily
Authors: Adermann Knut , Wattler Frank , Wattler Sigrid , Heine Gabriele , Meyer Markus , Forssmann Wolf‐Georg , Nehls Michael
Published: October 2010

Members of the Ly‐6/uPAR protein family share one or several repeat units of the Ly‐6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly‐6/uPAR protein family can be divided into two subfamilies. One comprises GPI‐anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single‐domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI‐anchoring signal sequence. We report the purification and structural characterization of human SLURP‐1 (secreted mammalian Ly‐6/uPAR related protein 1) from blood and urine peptide libraries. SLURP‐1 is encoded by the ARS (component B)‐81/s locus, and appears to be the first mammalian member of the Ly‐6/uPAR family lacking a GPI‐anchoring signal sequence. A phylogenetic analysis based on the SLURP‐1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP‐1 gene maps to the same chromosomal region as several members of the Ly‐6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co‐evolved from local chromosomal duplication events.
The compact and expanded denatured conformations of apomyoglobin in the methanol‐water solvent
Authors: Kamatari Yuji O. , Ohji Shoko , Konno Takashi , Seki Yasutaka , Soda Kunitsugu , Kataoka Mikio , Akasaka Kazuyuki
Published: October 2010

We have performed a detailed study of methanol‐induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far‐ and nearultraviolet circular dichroism, NMR spectroscopy, and small‐angle X‐ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid‐denatured (UA), compact denatured (IM), and expanded helical denatured (H) states, and indicates that the compact denatured state (IM) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (UA and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt‐pH plane but also in the alcohol‐pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, α‐lactalbumin, β‐lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer‐simulated Kratky profiles also supports this structural feature of the H state.
A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation
Authors: Beckett Dorothy , Kovaleva Elena , Schatz Peter J.
Published: October 2010

The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl‐CoA carboxylase. Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross‐species biotinylation has been demonstrated in several systems. To define the minimal substrate requirements in BirA‐catalyzed biotinylation, we have measured the kinetics of modification of a 23‐residue peptide previously identified by combinatorial methods. Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23‐residue peptide are similar to those determined for BCCP. To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino‐ and carboxy‐terminal truncations of the 23‐mer. The results, determined by stopped‐flow fluorescence, allowed identification of a 14‐residue peptide as the minimum required sequence. Additional support was obtained using matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl‐5′‐adenylate intermediate and catalytic amounts of BirA. Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation. The specificity constant (kcat/Km) governing BirA‐catalyzed biotinylation of the 14‐mer minimal substrate is similar to that determined for the natural substrate, BCCP. We conclude that the 14‐mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA.
Interaction with magnesium and ADP stabilizes both components of nitrogenase fromKlebsiella pneumoniaeagainst urea denaturation
Authors: Zou Lee , Baguinon Marilyn C. , Guo Xiaolu , Guo Sherry Yi , Yu Yong , Davis Lawrence C.
Published: October 2010

The nitrogenase enzyme ofKlebsiella pneumoniaeconsists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild‐type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2and GDP. Stabilization by Mg+2is slight for the holo MoFe protein (from ∼1.5 to ∼2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifHgene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2alone, to ∼3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of thenifMgene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifHgene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild‐type enzyme in the absence of Mg+2but comparable stability in its presence, showing the importance of Mg+2in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild‐type even with Mg+2added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild‐type.
Anti‐HIV and anti‐tumor protein MAP30, a 30 kDa single‐strand type‐I RIP, shares similar secondary structure and β‐sheet topology with the A chain of ricin, a type‐II RIP
Authors: Wang Yun‐Xing , Jacob Jaison , Torchia Dennis A. , Wemgfteld Paul T. , Palmer Ira , Stahl Stephen J. , Kaufman Joshua D. , Huang Philip Lin , Huang Paul Lee , Lee‐Huang Sylvia
Published: October 2010

MAP30 is a 30 kDa single‐stranded, type‐I ribosome inactivating protein (RIP) possessing anti‐tumor and anti‐HIV activities. It binds both ribosomal RNA and the HIV‐1 long‐terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 1Jhnha coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and β‐sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well‐known type‐II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three‐dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three‐dimensional structure and other structural properties such as surface electrostatic potentials.
Common EF‐hand motifs in cholinesterases and neuroligins suggest a role for Ca2+ binding in cell surface associations
Authors: Tsigelny I. , Taylor P. , Bourne P.E. , Shindyalov I.N. , Südhof T.C.
Published: October 2010

Comparisons of protein sequence via cyclic training of Hidden Markov Models (HMMs) in conjunction with alignments of three‐dimensional structure, using the Combinatorial Extension (CE) algorithm, reveal two putative EF‐hand metal binding domains in acetylcholinesterase. Based on sequence similarity, putative EF‐hands are also predicted for the neuroligin family of cell surface proteins. These predictions are supported by experimental evidence. In the acetylcholinesterase crystal structure from Torpedo californica, the first putative EF‐hand region binds the Zn2+ found in the heavy metal replacement structure. Further, the interaction of neuroligin 1 with its cognate receptor neurexin depends on Ca2+. Thus, members of the α, β hydrolase fold family of proteins contain potential Ca2+ binding sites, which in some family members may be critical for heterologous cell associations.
The π‐helix translates structure into function
Authors: Weaver Todd M.
Published: October 2010

A search for the occurrence of the rare π‐helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the π‐helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight π‐helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the π‐helix has evolved to provide unique structural features within a variety of proteins.
Conformational substates in different crystal forms of the photoactive yellow protein—Correlation with theoretical and experimental flexibility
Authors: Van Aalten Daan M.F. , Joshua‐Tor Leemor , Crielaard Wim , Hellingwerf Klaas J.
Published: October 2010

The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies. Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies. These studies have shown that conformational changes during the photocycle are limited to the chromophore and its immediate environment. However, spectroscopic studies suggest the presence of large conformational changes in the protein. Here, we address this apparent discrepancy in two ways. First, we obtain a description of large concerted motions in the ground state of the yellow protein from NMR data and theoretical calculations. Second, we describe the high‐resolution structure of the yellow protein crystallized in a different space group. The structure of the yellow protein differs significantly between the two crystal forms. We show that these differences can be used to obtain a description of the flexibility of the protein that is consistent with the motions observed in solution.
Comparison of sequence profiles. Strategies for structural predictions using sequence information
Authors: Rychlewski Leszek , Li Weizhong , Jaroszewski Lukasz , Godzik Adam
Published: October 2010

Distant homologies between proteins are often discovered only after three‐dimensional structures of both proteins are solved. The sequence divergence for such proteins can be so large that simple comparison of their sequences fails to identify any similarity. New generation of sensitive alignment tools use averaged sequences of entire homologous families (profiles) to detect such homologies. Several algorithms, including the newest generation of BLAST algorithms and BASIC, an algorithm used in our group to assign fold predictions for proteins from several genomes, are compared to each other on the large set of structurally similar proteins with little sequence similarity. Proteins in the benchmark are classified according to the level of their similarity, which allows us to demonstrate that most of the improvement of the new algorithms is achieved for proteins with strong functional similarities, with almost no progress in recognizing distant fold similarities. It is also shown that details of profile calculation strongly influence its sensitivity in recognizing distant homologies. The most important choice is how to include information from diverging members of the family, avoiding generating false predictions, while accounting for entire sequence divergence within a family. PSI‐BLAST takes a conservative approach, deriving a profile from core members of the family, providing a solid improvement without almost any false predictions. BASIC strives for better sensitivity by increasing the weight of divergent family members and paying the price in lower reliability. A new FFAS algorithm introduced here uses a new procedure for profile generation that takes into account all the relations within the family and matches BASIC sensitivity with PSI‐BLAST like reliability.
Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor
Authors: Charles Robert S. T. , Padmanabhan K. , Arni R. V. , Padmanabhan K. P. , Tulinsky A.
Published: October 2010

The structure of tick anticoagulant peptide (TAP) has been determined by X‐ray crystallography at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP‐BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c‐axis with a diameter of about 45 Å. Individual TAP units interact in a head‐to‐tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP‐BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N‐terminal tetrapeptide and the first disulfide loop of TAP (Cys5T‐Cys15T) upon binding to Xa. The TyrlTOH atom of TAP moves 14.2 Å to interact with Asp 189 of the S1 specificity site, Arg3TCZ moves 5.0 Å with the guanidinium group forming a cation‐π‐electron complex in the S4 subsite of Xa, while Lys7TNZ differs in position by 10.6 Å in TAP‐BPTI and TAP‐Xa, all of which indicates a different pre‐Xa‐bound conformation for the N‐terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP‐BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side‐chain conformations showing significant differences.
Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor
Authors: Jorissen Robert N. , Treutlein Herbert R. , Burgess Antony W. , Epa V. Chandana , Garrett Thomas P. J. , Ward Colin W.
Published: October 2010

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin‐like growth factor‐1 (IGF‐1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right‐handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod‐like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF‐1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.
Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry
Authors: Whittal Randy M. , Ball Haydn L. , Cohen Fred E. , Burlingame Alma L. , Prusiner Stanley B. , Baldwin Michael A.
Published: October 2010

Electrospray ionization mass spectrometry (ESI‐MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI‐MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N‐terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI‐MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C‐terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KD's are <100 nm. n‐terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both ph's. cu2+ also binds to a peptide corresponding to the extreme N‐terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four‐octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.
Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution
Authors: Goda Shuichiro , Takano Kazufumi , Yutani Katsuhide , Yamagata Yuriko , Nagata Ryou , Akutsu Hideo , Maki Saori , Namba Keiichi
Published: October 2010

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far‐ultraviolet region of the hen egg lysozyme changed to those characteristic of a β‐structure from the native α‐helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X‐ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of ∼70 Å. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X‐ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 Å, a reflection that arises from the interstrand spacing in β‐sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.
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

Electrospray 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

Proline 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

Well‐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

The 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

The 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

The 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

The 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

The 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

We 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

Surveys 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

A 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

We 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

In 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

One‐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.
Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles
Authors: Kanaji Y. , Kishida M. , Watanabe Y. , Kawamura T. , Xie S. , Yamashita Y. , Sassa C. , Tsukamoto Y.
Published: October 2010

Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles were investigated. Under transmitted light, translucent (Wt) and opaque otoliths (Wo) were detected in juveniles collected from Wakasa Bay between July 2005 and April 2006, whereas only opaque otoliths (Go) were detected in Goto‐nada Sea individuals between May and June 2006. Three groups of juveniles were distinguished based on differences in hatch season, otolith size and growth history, and body morphometrics. As T. japonicus has different spawning seasons according to spawning grounds, each group was estimated to hatch in different waters. Juveniles with Wt otoliths were considered to have stayed in coastal habitat longer, as the hatch area was estimated to be near Wakasa Bay. Juveniles with Wo and Go otoliths appear to recruit to coastal waters at larger size, since their hatch areas were estimated to be far from each collection area. Larger otoliths of Wt were attributed to otolith accretion after the second growth flexion, which was observed only for Wt. Standard length of Wt fish at the second otolith growth flexion was estimated to correspond to recruitment size to coastal rocky reefs in Wakasa Bay. Body morphometrics were correlated with otolith size after removing body size effect, suggesting that morphological variations of T. japonicus juveniles were also associated with the timing of recruitment to coastal habitat.