bluuebunny/crossref_metadata_2025_split huggingface dataset:
A natural variant of type I antifreeze protein with four ice‐binding repeats is a particularly potent antifreeze
Authors: Chao Heman , Hodges Robert S. , Kay Cyril M. , Gauthier Sherry Y. , Davies Peter L. 
Published: July 2010

AbstractA 4.3‐kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed‐phase HPLC. By the criteria of mass, amino acid composition, and N‐terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11‐amino acid repeats, one more than the major serum AFP components. The larger protein is completely α‐helical at 0 °C, with a melting temperature of 18 °C. It is considerably more active as an antifreeze than the three‐repeat winter flounder AFP and the four‐repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four‐repeat winter flounder AFP, including its larger size, additional ice‐binding residues, and differences in ice‐binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.
Microheterogeneity of human filaggrin: Analysis of a complex peptide mixture using mass spectrometry
Authors: Thulin Craig D. , Taylor J. Alex , Walsh Kenneth A. 
Published: July 2010

AbstractFilaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo.In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix‐assisted laser desorption time‐of‐flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.
Preliminary crystallographic analysis of the oxidized form of a two mono‐nuclear iron centres protein from desulfovibrio desulfuricans ATCC 27774
Authors: Coelho Ana V. , Matias Pedro M. , Carrondo Maria A. , Tavares P. , Moura J. J. G. , Moura Isabel , Fülop V. , Hajdu J. , Le Gall Jean 
Published: July 2010

AbstractCrystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 Å and c = 63.2 Å; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 Å, b = 80.9 Å, c = 53.9 Å, and β =98.1°. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self‐rotation function.
Subcloning of a dna fragment encoding a single cohesin domain of the clostridium thermocellum cellulosome‐integrating protein cipA: Purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide
Authors: Béguin Pierre , Raynaud Odette , Chaveroche Marie‐Kim , Dridi Abel , Alzari Pedro M. 
Published: July 2010

AbstractAn Escherichia coli clone encoding a single cohesin domain of the cellulosome‐integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P212121, with cell dimensions a = 37.7 Å, b = 80.7 Å, c = 93.3 Å, and four or eight molecules in the unit cell. The crystals diffract X‐rays to beyond 2 Å resolution and are suitable for further crystallographic studies.
Friends and relations of the cystatin superfamily—new members and their evolution
Authors: Brown W.M. , Dziegielewska K.M. 
Published: July 2010

AbstractThe cystatin “superfamily” encompasses proteins that contain multiple cystatin‐like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never aquired this inhibitory activity. In recent years, several new members of the superfamily have been characterized, including proteins from insects and plants. Based on partial amino acid homology, new members, such as the invariant chain (Ii), and the transforming growth factor‐β receptor type II (TGF‐β receptor II) may, in fact, represent members of an emerging family within the superfamily that may have used some common building blocks to form functionally diverse proteins. Cystatin super‐family members have been found throughout evolution and members of each family of the superfamily are present in mammals today. In this review, the new and older, established members of the family are arranged into a possible evolutionary order, based on sequence homology and functional similarities.
Structure of the β2 homodimer of bacterial luciferase from vibrio harveyi: X‐ray analysis of a kinetic protein folding trap
Authors: Thoden James B. , Holden Hazel M. , Fisher Andrew J. , Sinclair James F. , Wesenberg Gary , Baldwin Thomas O. , Rayment Ivan 
Published: July 2010

AbstractLuciferase, as isolated from Vibrio harveyi, is an aβ heterodimer. When allowed to fold in the absence of the α subunit, either in vitro or in vivo, the β subunit of the enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 °C. This form of the β subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320–326). Here we describe the X‐ray crystal structure of the β2 homodimer of luciferase from V. harveyi determined and refined at 1.95 Å resolution. Crystals employed in the investigation belonged to the orthorhombic space group P212121 with unit cell dimensions of a = 58.8 Å, b = 62.0 Å, and c = 218.2 Å and contained one dimer per asymmetric unit. Like that observed in the functional luciferase αβ heterodimer, the major tertiary structural motif of each β subunit consists of an (α/β)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three‐dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 Å resolution. Biochemistry 34: 6581–6586). The root‐mean‐square deviation of the α‐carbon coordinates between the β subunits of the hetero‐ and homodimers is 0.7 Å. This high resolution X‐ray analysis demonstrates that “domain” or “loop” swapping has not occurred upon formation of the β2 homodimer and thus the stability of the β2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the β2 homodimer and αβ heterodimer are remarkably similar in hydrogen‐bonding patterns and buried surface areas.
Molecular modeling of the structures of human and rat pancreatic cholesterol esterases
Authors: Feaster Shawn R. , Quinn Daniel M. , Barnett Bobby L. 
Published: July 2010

AbstractStructural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X‐ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contacts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of ϕ/Ψ angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the Cα coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model.A model for human cholesterol esterase was produced by repeating steps 1–3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.
Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies
Authors: Speed Margaret A. , Morshead Timothy , Wang Daniel I.C. , King Jonathan 
Published: July 2010

AbstractThe partitioning of partially folded polypeptide chains between correctly folded native states and off‐pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (Speed MA, Wang DIC, King J. 1995. Protein Sci 4:900–908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi‐Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945–15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off‐pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N‐terminus, indicating that the N‐terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N‐terminal epitope did not, indicating that the conformation of the N‐terminus is not a key determinant of the productive folding and chain association pathway.
Crystal structure of the reduced Schiff‐base intermediate complex of transaldolase B from escherichia coli: Mechanistic implications for class I aldolases
Authors: Jia Jia , Lindqvist Ylva , Schneider Gunter , Schörken Ulrich , Sprenger Georg A. 
Published: July 2010

AbstractTransaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff‐base intermediate between dihydroxyacetone and the ϵ‐amino group of a lysine residue at the active site of the enzyme is formed. This Schiff‐base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 Å resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.
Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue
Authors: Friedland Diana E. , Goss Dixie J. , Shoemaker Michael T. , Xie Yiping , Wang Yuhuan , Hagedorn Curt H. 
Published: July 2010

AbstractBinding of eIF‐4E to the 5′ m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the moiecular basis for this recognition, photoaffinity labeling with [γ‐32P]8‐N3GTP was used in binding site studies of human recombinant cap binding protein reIF‐4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [γ‐32P]8‐N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)‐chelate chromatography and reverse‐phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled reIF‐4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113‐Arg 122. Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [γ‐32P]8‐N3GTP. A detailed understanding of eIF‐4E‐m7G mRNA cap interactions may lead the way to regulating this essential protein‐RNA interaction for specific mRNA in vivo.
3‐Hydroxy‐3‐methylglutaryl‐coenzyme A reductase of haloferax volcanii: Role of histidine 398 and attenuation of activity by introduction of negative charge at position 404
Authors: Bischoff Kenneth M. , Rodwell Victor W. 
Published: July 2010

AbstractMutant 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG‐CoA reductases via charge‐charge interaction with the active site histidine. To first verify the sequence‐based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG‐CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG‐CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403‐residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii‐hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild‐type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.
γ‐Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function
Authors: Gillis Shmuel , Furie Barbara C. , Furie Bruce , Patel Himakshi , Huberty Michael C. , Switzer Mary , Barry Foster W. , Scoble Hubert A. , Bond Michael D. 
Published: July 2010

AbstractThe γ‐carboxyglutamic acid (Gla) domains of the vitamin K‐dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion‐exchange chromatography, peptide mapping, mass spectrometry, and N‐terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/γ40E is uncarboxylated at residue 40 and factor IX/γ36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully y‐carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma‐derived factor IX. As monitored by anti‐factor IX:Ca(II)‐specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)‐induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half‐maximal inhibition of the binding of 125I‐labeled plasma‐derived factor IX at concentrations of 2–6 nM. Functionally, factor IX/γ36,40E and factor IX/γ40E were similar to fully γ‐carboxylated recombinant factor IX and plasma‐derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti‐factor IX:Mg(II)‐specific antibodies because these antibodies bound factor IX preferentially to factor LX/γ36,40E and factor IX/γ40E. These results demonstrate that the γ‐carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.
Mechanism of the stabilization of ribonuclease a by sorbitol: Preferential hydration is greater for the denatured than for the native protein
Authors: Xie Guifu , Timasheff Serge N. 
Published: July 2010

AbstractThe effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, δΔG° = ΔG°(sorbitol) ‐ ΔG°(water). Measurements of preferential interactions at 48 °C at pH 5.5, where protein is native, and pH 2.0, where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, ΔμN

2 > Δμ
N

2, which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co‐solvent on denaturation, ΔG°

w + Δμ
D
2 = ΔG°
S + Δμ
D

2 indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water co‐solvent exchange at thermodynamically non‐neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.
Temperature dependence of the preferential interactions of ribonuclease a in aqueous co‐solvent systems: Thermodynamic analysis
Authors: Xie Guifu , Timasheff Serge N. 
Published: July 2010

AbstractThe temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co‐solvents, more so at low pH than high pH (except 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co‐solvents was high at temperatures below 30 °C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 °C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (∂μ2/∂m3)T, P, m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, \documentclass{article}\pagestyle{empty}\begin{document}$ \Delta \bar H_{2,tr} $\end{document}, and transfer entropy \documentclass{article}\pagestyle{empty}\begin{document}$ \Delta \bar S_{2,tr} $\end{document}. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water to a 30% sorbitol solution.
A new method for analyzing protein sequence relationships based on Sammon maps
Authors: Agrafiotis Dimitris K. 
Published: July 2010

AbstractRecent advances in gene sequencing and rational drug design have re‐emphasized the need for new methods for protein analysis, classification, and structure and function prediction. In this article, we introduce a new method for analyzing protein sequences based on Sammon's non‐linear mapping algorithm. When applied to a family of homologous sequences, the method is able to capture the essential features of the similarity matrix, and provides a faithful representation of chemical or evolutionary distance in a simple and intuitive way. The merits of the new algorithm are demonstrated using examples from the protein kinase family.