bluuebunny/crossref_metadata_2025_split huggingface dataset:
A peptide analog of the calmodulin‐binding domain of myosin light chain kinase adopts an aL‐helical structure in aqueous trifluoroethanol
Authors: Zhang Mingjie , Yuan Tao , Vogel Hans J. 
Published: February 2009

AbstractA 22‐residue synthetic peptide encompassing the calmodulin (CaM)‐binding domain of skeletal muscle myosin light chain kinase was studied by two‐dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix‐inducing solvent trifluoroethanol (TFE) causes it to form an α‐helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum‐filtered J‐correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the α‐carbon chemical shifts were obtained from (1H,13C)‐heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), dαN( i, i + 3), and d αβ (i, i + 3) NOE crosspeaks indicates that an α ‐helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield α ‐proton and downfield α ‐carbon shifts in this region of the peptide provide further support for the formation of an α ‐helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.
A 19F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli
Authors: Sun Zhen‐Yu , Truong Hoai‐Thu N. , Pratt E. A. , Sutherland David C. , Kulig Christine E. , Homer Ronald J. , Groetsch Stefan M. , Hsue Priscilla Y. , Ho Chien 
Published: February 2009

Abstractd‐Lactate dehydrogenase (d‐LDH) is a membrane‐associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65, 000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of d‐LDH and its interaction with phospholipids. We incorporated 5‐fluorotryptophan (5F‐Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide‐directed mutagenesis, and studied the 5F‐Trp‐labeled enzymes using 19F‐NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin‐labeled fatty acid, which broadens the resonance from any residue within 15 Å, we have established that the membrane‐binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F‐NMR spectroscopy of wild‐type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin‐labeled fatty acid. These experiments indicate that 9–10 Phe and 3–4 Tyr residues are located near the lipid phase.
Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation
Authors: Thomas Paul D. , Dill Ken A. 
Published: February 2009

AbstractHow important are helical propensities in determining the conformations of globular proteins? Using the two‐dimensional lattice model and two monomer types, H (hydrophobic) and P (polar), we explore both nonlocal interactions, through an HH contact energy, as developed in earlier work, and local interactions, through a helix energy, σ. By computer enumeration, the partition functions for short chains are obtained without approximation for the full range of both types of energy. When nonlocal interactions dominate, some sequences undergo coil‐globule collapse to a unique native structure. When local interactions dominate, all sequences undergo helix–coil transitions. For two different conformational properties, the closest correspondence between the lattice model and proteins in the Protein Data Bank is obtained if the model local interactions are made small compared to the HH contact interaction, suggesting that helical propensities may be only weak determinants of globular protein structures in water. For some HP sequences, varying σ/ leads to additional sharp transitions (sometimes several) and to “conformational switching” between unique conformations. This behavior resembles the transitions of globular proteins in water to helical states in alcohols. In particular, comparison with experiments shows that whereas urea as a denaturant is best modeled as weakening both local and nonlocal interactions, trifluoroethanol is best modeled as mainly weakening HH interactions and slightly enhancing local helical interactions.
Identification of the amino acids comprising a surface‐exposed epitope within the nucleotide‐binding domain of the na+, K+‐ATPase using a random peptide library
Authors: Malik Bela , Jamieson Gordon A. , Ball W. James 
Published: February 2009

AbstractMonoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8‐P1‐A3 has been shown to bind to the catalytic (α) subunit of the Na+, K+‐ATPase holoenzyme and the synthetic peptide sequence 496‐HLLVMK*GAPER‐506, which includes Lys 501 (K*), the major site for fluorescein‐5′‐isothio‐cyanate labeling of the Na+, K+‐ATPase. This sequence region of α is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W.R. & Green, N.W., 1989, Eur. J. Biochem. 179, 241–248). In this study we have determined M8‐P1‐A3′s ability to recognize the α‐subunit or homologous E1E2‐ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or “epitope” library, recently developed by Scott and Smith (1990, Science 249, 386–390) and Devlin et al. (Devlin, J.J., Panganiban, L.C., & Devlin, P.E., 1990, Science 249, 404–406), has served as a convenient technique to confirm the species‐specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8‐P1‐A3 epitope was found to consist of the five amino acid 494–PRHLL‐498 sequence stretch of α, with residues PRxLx being critical for antibody recognition.
Origins of structural diversity within sequentially identical hexapeptides
Authors: Cohen Bruce I. , Presnell Scott R. , Cohen Fred E. 
Published: February 2009

AbstractEfforts to predict protein secondary structure have been hampered by the apparent structural plasticity of local amino acid sequences. Kabsch and Sander (1984, Proc. Natl. Acad. Sci. USA 81, 1075–1078) articulated this problem by demonstrating that identical pentapeptide sequences can adopt distinct structures in different proteins. With the increased size of the protein structure database and the availability of new methods to characterize structural environments, we revisit this observation of structural plasticity. Within a set of proteins with less than 50% sequence identity, 59 pairs of identical hexapeptide sequences were identified. These local structures were compared and their surrounding structural environments examined. Within a protein structural class (α/α, β/β, α/β, α + β), the structural similarity of sequentially identical hexapeptides usually is preserved. This study finds eight pairs of identical hexapeptide sequences that adopt β‐strand structure in one protein and α‐helical structure in the other. In none of the eight cases do the members of these sequence pairs come from proteins within the same folding class. These results have implications for class dependent secondary structure prediction algorithms.
Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability
Authors: Pjura Philip , Matsumura Masazumi , Baase Walter A. , Matthews Brian W. 
Published: February 2009

AbstractAn M13 bacteriophage‐based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates. Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one‐step mutagenesis and screening system has been used to find 18 random single‐site mutant lysozymes, of which 11 were heat resistant. Each of these had a melting temperature within 0.8–1.4°C of wild type, suggesting that the screening system is quite sensitive.
Side‐chain entropy and packing in proteins
Authors: Bromberg Sarina , Dill Ken A. 
Published: February 2009

AbstractWhat role does side‐chain packing play in protein stability and structure? To address this question, we compare a lattice model with side chains (SCM) to a linear lattice model without side chains (LCM). Self‐avoiding configurations are enumerated in 2 and 3 dimensions exhaustively for short chains and by Monte Carlo sampling for chains up to 50 main‐chain monomers long. This comparison shows that (1) side‐chain degrees of freedom increase the entropy of open conformations, but side‐chain steric exclusion decreases the entropy of compact conformations, thus producing a substantial entropy that opposes folding; (2) there is a side‐chain “freezing” or ordering, i.e., a sharp decrease in entropy, near maximum compactness; and (3) the different types of contacts among side chains (s) and main‐chain elements (m) have different frequencies, and the frequencies have different dependencies on compactness.mmcontacts contribute significantly only at high densities, suggesting that main‐chain hydrogen bonding in proteins may be promoted by compactness. The distributions ofmm, ms, andsscontacts in compact SCM configurations are similar to the distributions in protein structures in the Brookhaven Protein Data Bank. We propose that packing in proteins is more like the packing of nuts and bolts in a jar than like the pairwise matching of jigsaw puzzle pieces.
Relative stabilities of synthetic peptide homo‐ and heterodimeric troponin‐C domains
Authors: Shaw Gary S. , Hodges Robert S. , Kay Cyril M. , Sykes Brian D. 
Published: February 2009

AbstractIt has previously been shown that synthetic peptides corresponding to calcium‐binding sites III (SCIII) and IV (SCIV) from troponin‐C can undergo a calcium‐induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280–283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258–6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer (Shaw GS et al., 1992a, J Am Chem Soc 114:6258–6259). The stabilities of these dimers have been investigated by using 1H‐NMR and circular dichroism spectroscopies to follow temperature‐ and guanidine hydrochloride (GuHCl)‐induced denaturations. It has been found that the most stable species, the SCIII/SCIV heterodimer (δGu = –64.8 kJ/mol), is about 13 kJ/mol more stable than the least stable species, the SCIV homodimer, while the SCIII homodimer is of intermediate stability. This trend of free energies agrees well with the trend of δG0 values derived from the products of the dissociation constants for calcium binding and peptide association determined from earlier calcium‐titration studies. These observations provide evidence that calcium affinity and the association of 2‐calcium binding sites are tightly linked. However, it was noted that in all cases δG0 was considerably more negative than δGu determined from GuHCl experiments. This difference increased as the stability of the peptide complex increased, providing evidence that linear extrapolation of GuHCl data for very stable proteins may significantly underestimate the value for δG0.
Interaction of the DNA‐binding domain of Drosophila heat shock factor with its cognate DNA site: A thermodynamic analysis using analytical ultracentrifugation
Authors: Kim Soon‐Jong , Tsukiyama Toshio , Lewis Marc S. , Wu Carl 
Published: February 2009

AbstractHeat shock transcription factor (HSF) mediates the activation of heat shock genes by binding to its cognate sites with high affinity and specificity. The high‐affinity binding of HSF is dependent on the formation of an HSF homotrimer, which interacts specifically with the heat shock response element (HSE), comprised of 3 inverted repeats of the 5‐bp sequence NGAAN. In order to investigate the thermodynamic basis of the interaction between HSF and HSE, we have overexpressed and purified a polypeptide (dHSF(33–163)) encompassing only the DNA‐binding domain of HSF from Drosophila and analyzed its binding to DNA by equilibrium analytical ultracentrifugation using a multiwavelength scan technique. We demonstrate that dHSF(33–163) can bind as a monomer with 1:1 stoichiometry to a synthetic 13‐bp DNA containing a single NGAAN sequence. The values of the thermodynamic parameters obtained from the temperature dependence of the equilibrium binding constants indicate that the changes of free energy for the binding of dHSF(33–163) to the wild‐type site and a mutant DNA site are predominantly characterized by substantial negative changes of enthalpy. Binding to the wild‐type DNA is characterized by a significant positive change of entropy, whereas binding to the mutant DNA is distinguished by a negative change of entropy of comparable magnitude. The binding to the mutant DNA was also highly sensitive to increasing salt concentrations, indicating a dominance of ionic interactions. The sequence‐specific, 1:1 binding of dHSF(33–163) to the NGAAN sequence provides a basis for the analysis of higher order interactions between HSF trimers and the HSE.
UDP‐glucose dehydrogenase from bovine liver: Primary structure and relationship to other dehydrogenases
Authors: Hempel John , Perozich John , Romovacek Hana , Hinich Amy , Kuo Ingrid , Feingold David Sidney 
Published: February 2009

AbstractThe primary structure of bovine liver UDP‐glucose dehydrogenase (UDPGDH), a hexameric, NAD+‐linked enzyme, has been determined at the protein level. The 52‐kDa subunits are composed of 468 amino acid residues, with a free N‐terminus and a Ser/Asn microheterogeneity at one position. The sequence shares 29.6% positional identity with GDP‐mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to “UDP‐N‐acetyl‐mannosaminuronic acid dehydrogenase” from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of ∼26–34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4‐electron‐transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half‐reaction (conversion of UDP‐aldehydoglucose to UDP‐glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half‐reaction (conversion of UDP‐glucose to UDP‐aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme‐binding fold is found at positions 11–16, close to the N‐terminus as with “short‐chain” alcohol dehydrogenases. Because the enzyme combines functionalities of alcohol and aldehyde dehydrogenases, it was also of interest to search specifically for other sequence similarities to either of these 2 enzymes, as well as to histidinol dehydrogenase, another 4‐electron‐transferring dehydrogenase, but none were found.
Crystal structure of inorganic pyrophosphatase from Thermus thermophilus
Authors: Teplyakov Alexei , Obmolova Galya , Wilson Keith S. , Ishii Keisuke , Kaji Hiroyuki , Samejima Tatsuya , Kuranova Inna 
Published: February 2009

AbstractThe 3‐dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T‐PPase) has been determined by X‐ray diffraction at 2.0 Å resolution and refined to R = 15.3%. The structure consists of an antiparallel closed β‐sheet and 2 α‐helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek‐key topology of the 5‐stranded β‐barrel was found in the OB‐fold proteins, the bacteriophage gene‐5 DNA‐binding protein, toxic‐shock syndrome toxin‐1, and the major cold‐shock protein of Bacillus subtilis. Moreover, all known nucleotide‐binding sites in these proteins are located on the same side of the β‐barrel as the active center in T‐PPase. Analysis of the active center of T‐PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site‐directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T‐PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.
The pharmacophore of the human C5a anaphylatoxin
Authors: Toth Matthew J. , Huwyler Leslie , Boyar William C. , Braunwalder Albert F. , Yarwood Donna , Hadala Joseph , Haston William O. , Sills Matthew A. , Seligmann Bruce , Galakatos Nicholas 
Published: February 2009

AbstractWe have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74‐amino acid protein was performed and the effects on the potency of receptor binding and of C5a‐induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site‐directed mutagenesis. Ten noncontiguous amino acids from the structurally well‐defined 4‐helix core domain (amino acids 1–63) and the C‐terminal arginine‐containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C‐terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.
Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals
Authors: Duke E.M.H. , Wakatsuki S. , Hadfield A. , Johnson L.N. 
Published: February 2009

AbstractThe conversion of substrate, heptenitol, to product, β‐1‐C‐methyl, α‐D‐glucose‐1‐phosphate (heptulose‐2‐P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5‐dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer.In the Laue diffraction experiments, data to 2.8 Å resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800‐ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed.Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 Å resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose‐2‐P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 Å resolution to establish the fractional occupancies of the enzyme‐substrate and enzyme‐product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal. In these experiments in which only partial reaction had occurred, there was connecting density between the phosphate and the O2 hydroxyl of the sugar substrate. The recent evidence for the influence of the O2 hydroxyl on the catalytic mechanism is discussed.
Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala
Authors: De Llano Jose Javier Martin , Manning James M. 
Published: February 2009

AbstractA recombinant double mutant of hemoglobin (Hb), E6V/L88A(β), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val‐6(β) of one tetramer and the hydrophobic region between Phe‐85(β) and Leu‐88(β) on an adjacent tetramer. Thus, a construct encoding the donor Val‐6(β) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu‐88(β) was assembled. The doubly mutated β‐globin gene was expressed in yeast together with the normal human α‐chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant β‐chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor‐acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.