bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alignment of 700 globin sequences: Extent of amino acid substitution and its correlation with variation in volume
Authors: Kapp Oscar H. , Moens Luc , Vanfleteren Jaak , Trotman Clive N.A. , Suzuki Tomohiko , Vinogradov Serge N. 
Published: February 2009

AbstractSeven‐hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation ∼2–3%, the variation in volume per position ranges from ∼13% for the internal to ∼21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pair‐wise similarity scores. The scores for globin‐globin comparisons over the 84 common positions overlap the globin‐phycocyanin and globin‐colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three‐on‐three α‐helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.
Side‐chain conformational entropy in protein folding
Authors: Doig Andrew J. , Sternberg Michael J. E. 
Published: February 2009

AbstractAn important, but often neglected, contribution to the thermodynamics of protein folding is the loss of entropy that results from restricting the number of accessible side‐chain conformers in the native structure. Conformational entropy changes can be found by comparing the number of accessible rotamers in the unfolded and folded states, or by estimating fusion entropies. Comparison of several sets of results using different techniques shows that the mean conformational free energy change (TΔS) is 1 kcal‐mol−1 per side chain or 0.5 kcal‐mol−1 per bond. Changes in vibrational entropy appear to be negligible compared to the entropy change resulting from the loss of accessible rotamers. Side‐chain entropies can help rationalize α‐helix propensities, predict protein/inhibitor complex structures, and account for the distribution of side chains on the protein surface or interior.
Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus)
Authors: Itoh Susumu , Navia Manuel A. 
Published: February 2009

AbstractThe consequences of site‐directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of “conservative” and “nonconservative” substitutions on the X‐ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110‐fold and 180‐fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506‐mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so‐called “40s loop” that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42‐mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.
Modeling of the structure of the Haemophilus influenzae heme‐binding protein suggests a mode of heme interaction
Authors: Dunten Pete , Mowbray Sherry L. 
Published: February 2009

AbstractThe structure and function of the periplasmic heme‐binding protein HbpA of Haemophilus influenzae were investigated. This protein is involved in the import of heme into the bacteria through the inner membrane, and thus is a key element of the organism's ability to survive in blood. A high degree of sequence similarity between HbpA and the dipeptide‐binding protein of Escherichia coli is suggested to be the result of a functional relationship. An HbpA model built using the dipeptide‐binding protein suggests a mode of heme binding that is distinct from those known in proteins of the human host. These results provide a starting point for rational drug design.
Homology modeling and molecular dynamics simulation of human prothrombin fragment 1
Authors: Li Leping , Darden Tom , Foley Charles , Hiskey Richard , Pedersen Lee 
Published: February 2009

AbstractThe crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hf1/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long‐range ionic forces was essential to reach a stable solution structure. The γ‐carboxyglutamic acid (Gla) domain and the kringle domain of hf1/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K‐dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.
Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities
Authors: Toney Michael D. , Pascarella Stefano , De Biase Daniela 
Published: February 2009

AbstractA homology model for the pig isozyme of the pyridoxal phosphate‐dependent enzyme γ‐aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the pro‐S proton at C4 of GABA, which abstracted in the 1,3‐azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic ω‐amino acids such as GABA. In the proposed structure for the L‐glutamate external aldimine, the α‐carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on ω‐amino acids in one half‐reaction and on α‐amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of β‐substituted phenyl and p‐chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism‐based inhibitor vinyl‐GABA, an effective anti‐epileptic drug known clinically as Vigabatrin, would orient the scissile C4‐H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4‐H bond toward Arg 445. The active site model presented provides a basis for site‐directed mutagenesis and drug design experiments.
Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure
Authors: Kreisberg Racheli , Buchner Virginia , Arad Dorit 
Published: February 2009

AbstractThis paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural‐cysteine‐mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide‐bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three‐dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.
Crystallization and preliminary X‐ray analysis of fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase
Authors: Istvan Eva S. , Hasemann Charles A. , Kurumbail Ravi G. , Uyeda Kosaku , Deisenhofer Johann 
Published: February 2009

AbstractDiffraction‐quality crystals of the bifunctional enzyme fructose 6‐phosphate, 2‐kinase:fructose 2,6‐bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATPγS, fructose 6‐phosphate, the detergent n‐octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 Å and c = 130.6 Å. Flash‐frozen crystals diffract to beyond 2.2 Å, and native data have been collected.
N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose
Authors: Oikonomakos N. G. , Kontou M. , Zographos S. E. , Watson K. A. , Johnson L. N. , Bichard C. J. F. , Fleet G. W. J. , Acharya K. R. 
Published: February 2009

AbstractStructure‐based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α‐D‐glucose (Ki = 1.7 mM). The best inhibitor in the class of N‐acyl derivatives of β‐D‐glucopyranosylamine, N‐acetyl‐β‐D‐glucopyranosylamine (1‐GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1‐GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 μM) and the α (Ki = 35 μM) forms of the enzyme with respect to glucose 1‐phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1‐GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T‐state conformation. Co‐crystals of the phosphorylase b‐1‐GlcNAc‐IMP complex were grown in space group P43212, with native‐like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1‐GlcNAc binds tightly at the catalytic site of T‐state phosphorylase b at approximately the same position as that of α‐D‐glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T‐state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T‐state phosphorylase b‐1‐GlcNAc‐IMP complex structure is overall similar to the T‐state phosphorylase b‐α‐D‐glucose complex structure. The structure of the 1‐GlcNAc complex provides a rational for the biochemical properties of the inhibitor.
Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects
Authors: Assemat Karine , Clément‐Métral Jenny , Alzari Pedro M. 
Published: February 2009

AbstractThe internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, IIe, Tyr) by site‐directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA− background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45‐60% that of the wild type at 37°, and essentially identical at 42°. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37°, demonstrating that subtle modifications of the protein interior at position 25 (Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro.
Chemically crosslinked protein dimers: Stability and denaturation effects
Authors: Byrne Michael P. , Stites Wesley E. 
Published: February 2009

AbstractNine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1, 6‐bismaleimidohexane (BMH), 1, 3‐dibromo‐2‐propanol (DBP), and the chemical warfare agent, mustard gas (bis(2‐chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two‐state model of unfolding. The monofunctional reagent, e‐maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three‐state thermodynamic model of two successive transitions in which the individual sub‐units cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two‐state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.
Significant discrepancies between van't hoff and calorimetric enthalpies. II
Authors: Liu Yufeng , Sturtevant Julian M. 
Published: February 2009

AbstractIsothermal calorimetric titration of 18‐crown‐6 ether with BaCl2 in pure aqueous solution over the temperature range 7‐40 °C gives precise binding constants and enthalpy changes. Nonlinear least‐squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature‐independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.
The role of a conserved tyrosine residue in high‐potential iron sulfur proteins
Authors: Iwagami Sakura G. , Creagh A. Louise , Haynes Charles A. , Borsari Marco , Felli Isabella C. , Piccioli Mario , Eltis Lindsay D. 
Published: February 2009

AbstractConserved tyrosine‐12 of Ectothiorhodospira halophila high‐potential iron sulphur protein (HiPIP) iso‐I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild‐type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0‐11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr‐12 hydroxyl group with Asn‐14 Oδ1 and Lys‐59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys‐59/Val‐58 peptide bond, thereby perturbing Gly‐46. The ΔΔGappD of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 °C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr‐12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 °C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr‐12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.
Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy
Authors: Frank M. Kirsten , Clore G. Marius , Gronenborn Angela M. 
Published: February 2009

AbstractThe structure and dynamics of the urea‐denatured B1 immunoglobulin binding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 1H, 15N, and 13C assignments are obtained by means of sequential through‐bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHNα coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. 15N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second β‐hairpin, the N‐terminus of the α‐helix, and the middle of the α‐helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (∼3 ns) and could range from about 6 ns in the case of one model to between 4 μs and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N‐ and C‐termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5.
Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences
Authors: Bayley P.M. , Findlay W.A. , Martin S.R. 
Published: February 2009

AbstractThe interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N‐terminal target residues and the C‐domain of calmodulin, and between the C‐terminal target residues and the N‐domain of calmodulin (Ikura M et al., 1992, Science 256:632–638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C‐domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1–10 of M13) binds to CaM with Kd ≈︁ 1 μM; peptide FW10 (residues 9–18 of M13, with Phe‐17 → Trp substitution) binds to CaM with Kd ≈︁ 100 μM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C‐domain, forming a stable half‐saturated complex, Ca2‐CaM‐peptide, and confirming the functional importance of the interaction of this sequence with the C‐domain. Stopped‐flow studies show that the EGTA‐induced dissociation of WF10 from Ca4‐CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half‐saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N‐terminal target residues with the C‐domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N‐ and C‐domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin‐dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C‐domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.