bluuebunny/crossref_metadata_2025_split huggingface dataset:
Escherichia coli alkaline phosphatase: X‐ray structural studies of a mutant enzyme (His‐412 → Asn) at one of the catalytically important zinc binding sites
Authors: Ma Lan , Tibbitts Thomas T. , Kantrowitz Evan R. 
Published: February 2009

AbstractThe X‐ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His‐412 was replaced by Asn has been determined at both low (‐Zn) and high (+Zn) concentrations of zinc. In the wild‐type structure, His‐412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn,) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614–31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild‐type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N(+Zn) enzyme was refined to an R‐factor of 0.174 at 2.2 Å resolution, and the structure of the H412N(‐Zn) enzyme was refined to an R‐factor of 0.166 at a resolution of 2.6 Å. Both indicated that the Asn residue substituted for His‐412 did not coordinate well to Zn1. In the H412N(‐Zn) structure, the Zn, site had very low occupancy and the phosphate was shifted by 1.8 Å from its position in the wild‐type structure. The Mg binding site was also affected by the substitution of Asn for His‐412. Both structures of the H412N enzyme also revealed a surface‐accessible cavity near the Zn, site that may serve as a binding site for Tris. The binding of Tris at the Zn, site may block Zn2+ binding at this site, which would account for the reduced affinity of the H412N enzyme for substrate as well as its reduced catalytic efficiency in the presence of Tris. Thus, the replacement of His‐412 with Asn affects all three metal binding sites, suggesting that the relative positions of the metals bound at the active site of E. coli alkaline phosphatase are strongly linked and critical for efficient catalysis.
The optimization of protein‐solvent interactions: Thermostability and the role of hydrophobic and electrostatic interactions
Authors: Spassov Velin Z. , Karshikoff Andrej D. , Ladenstein Rudolf 
Published: February 2009

AbstractProtein‐solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent‐accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three‐dimensional structure available in the Protein Data Bank. The dependence of the total solvent‐accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent‐accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein‐solvent and charge‐charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge‐charge interactions.
Control of aggregation in protein refolding: A variety of surfactants promote renaturation of carbonic anhydrase II
Authors: Wetlaufer D.B. , Xie Y. 
Published: February 2009

AbstractThe denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of ∼0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle‐forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.
Gibbs motif sampling: Detection of bacterial outer membrane protein repeats
Authors: Neuwald Andrew F. , Liu Jun S. , Lawrence Charles E. 
Published: February 2009

AbstractThe detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif‐encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix‐turn‐helix DNA‐binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403–410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric β‐barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane‐spanning β‐strands. These β‐strands occur on the membrane interface (as opposed to the trimeric interface) of the β‐barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles.
Pseudotorsional OCCO backbone angle as a single descriptor of protein secondary structure
Authors: Laiter Sergei , Vaisman Iosif I. , Tropsha Alexander , Hoffman Doug L. , Singh Raj K. 
Published: February 2009

AbstractProtein secondary structure is conventionally identified using characteristic ranges of two backbone torsional angles π and Ψ We suggest that the secondary structure can be adequately characterized by a single descriptor, the Oi‐1 Ci‐1CiOi (where i is the residue number) pseudotorsional backbone angle. A set of 102 structurally distinct protein chains from the Protein Data Bank was used to evaluate the adequacy of this descriptor. We find that a specific range of OCCO angles corresponds to each major secondary structure. The complete range of OCCO angles (‐180° to 179°) was broken into 18 consecutive subranges of 20° each, and each subrange was assigned a letter. Thus, the OCCO profiles for each protein in the database were “translated” into a sequence of letters. The Needleman‐Wunsch primary sequence alignment algorithm was then used for secondary/tertiary structure comparison and alignment. Preliminary results indicate that this new approach has a significant potential for rapid identification of fold families in the Protein Data Bank.
Crystallization and preliminary X‐ray analysis of electron transfer flavoproteins from human and paracoccus denitrificans
Authors: Roberts David L. , Kim Jung‐Ja P. , Herrick Kevin R. , Frerman Frank E. 
Published: February 2009

AbstractMammalian electron transfer flavoprotein (ETF) is a soluble, heterodimeric flavoprotein responsible for the oxidation of at least nine primary matrix flavoprotein dehydrogenases. Crystals have been obtained for the recombinant human electron transfer flavoprotein (ETFhum) by the sitting‐drop vapor diffusion technique using polyethylene glycol (PEG) 1500 at pH 7.0 as the precipitating agent. ETFhum crystallizes in the monoclinic space group P21, with unit cell parameters a = 47.46 Å, b = 104.10 Å, c = 63.79 Å, and β = 110.02°. Based on the assumption of one αβ dimer per asymmetric unit, the Vm value is 2.69 Å3/Da. A native data set has been collected to 2.1 Å resolution. One heavy‐atom derivative has also been obtained by soaking a preformed crystal of ETFhum in 2 mM thimerosal solution for 2 h at 19 °C. Patterson analysis indicates one major site. The analogous electron transfer flavoprotein from Paracoccus denitrificans (ETFpar) has also been crystallized using PEG 8000 at pH 5.5 as the precipitating agent. ETFpar crystallizes in the orthorhombic space group P212121, with unit cell parameters a = 79.98 Å, b = 182.90 Å, and c = 70.07 Å. The Vm value of 2.33 Å3/Da is consistent with two αβ dimers per asymmetric unit. A native data set has been collected to 2.5 Å resolution.
Crystal structure of thioltransferase at 2.2 Å resolution
Authors: Katti Suresh K. , Robbins Arthur H. , Yang Yanfeng , Wells William W. 
Published: February 2009

AbstractWe report here the first three‐dimensional structure of a mammalian thioltransferase as determined by single crystal X‐ray crystallography at 2.2 Å resolution. The protein is known for its thiol‐redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy‐atom derivatives. The protein folds into an α/β structure with a four‐stranded mixed β‐sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol‐redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22‐Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis‐peptide configuration. Mutational data available on the protein are in agreement with the three‐dimensional structure.
De novo design of the hydrophobic cores of proteins
Authors: Desjarlais John R. , Handel Tracy M. 
Published: February 2009

AbstractWe have developed and experimentally tested a novel computational approach for the de novo design of hydrophobic cores. A pair of computer programs has been written, the first of which creates a “custom” rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program uses a genetic algorithm to globally optimize for a low energy core sequence and structure, using the custom rotamer library as input. Success of the programs in predicting the sequences of native proteins indicates that they should be effective tools for protein design.Using these programs, we have designed and engineered several variants of the phage 434 cro protein, containing five, seven, or eight sequence changes in the hydrophobic core. As controls, we have produced a variant consisting of a randomly generated core with six sequence changes but equal volume relative to the native core and a variant with a “minimalist” core containing predominantly leucine residues. Two of the designs, including one with eight core sequence changes, have thermal stabilities comparable to the native protein, whereas the third design and the minimalist protein are significantly destabilized. The randomly designed control is completely unfolded under equivalent conditions. These results suggest that rational de novo design of hydrophobic cores is feasible, and stress the importance of specific packing interactions for the stability of proteins. A surprising aspect of the results is that all of the variants display highly cooperative thermal denaturation curves and reasonably dispersed NMR spectra. This suggests that the non‐core residues of a protein play a significant role in determining the uniqueness of the folded structure.
Prediction of polyelectrolyte polypeptide structures using Monte Carlo conformational search methods with implicit solvation modeling
Authors: Evans John Spencer , Chan Sunney I. , Goddard William A. 
Published: February 2009

AbstractMany interesting proteins possess defined sequence stretches containing negatively charged amino acids. At present, experimental methods (X‐ray crystallography, NMR) have failed to provide structural data for many of these sequence domains. We have applied the dihedral probability grid‐Monte Carlo (DPG‐MC) conformational search algorithm to a series of N‐ and C‐capped polyelectrolyte peptides, (Glu)20, (Asp)20. (PSer)20, and (PSer‐Asp)10, that represent polyanionic regions in a number of important proteins, such as parathymosin, calsequestrin, the sodium channel protein, and the acidic biomineralization proteins. The atomic charges were estimated from charge equilibration and the valence and van der Waals parameters are from DREIDING. Solvation of the carboxylate and phosphate groups was treated using sodium counterions for each charged side chain (one Na+ for COO−; two Na for CO(PO3)−2) plus a distance‐dependent (shielded) dielectric constant, ϵ = ϵ0R, to simulate solvent water. The structures of these polyelectrolyte polypeptides were obtained by the DPG‐MC conformational search with ϵ0 = 10, followed by calculation of solvation energies for the lowest energy conformers using the protein dipole‐Langevin dipole method of Warshel.These calculations predict a correlation between amino acid sequence and global folded conformational minima:
Poly‐L‐Glu20, our structural benchmark, exhibited a preference for right‐handed α‐helix (47% helicity), which approximates experimental observations of 55–60% helicity in solution.
For Asp‐ and PSer‐containing sequences, all conformers exhibited a low preference for right‐handed α‐helix formation (≤10%), but a significant percentage (?20% or greater) of β‐strand and β‐turn dihedrals were found in all three sequence cases: (1) Aspn forms supercoil conformers, with a 2:1:1 ratio of β‐turn:β‐strand:α‐helix dihedral angles; (2) PSer20 features a nearly 1:1 ratio of β‐turn:β‐sheet dihedral preferences, with very little preference for α‐helical structure, and possesses short regions of strand and turn combinations that give rise to a collapsed bend or hairpin structure; (3) (PSer‐Asp)10 features a 3:2:1 ratio of β‐sheet:β‐turn:α‐helix and gives rise to a superturn or C‐shaped structure.
Binary patterning of polar and nonpolar amino acids in the sequences and structures of native proteins
Authors: West Michael W. , Hecht Michael H. 
Published: February 2009

AbstractProtein sequences can be represented as binary patterns of polar (○) and nonpolar (•) amino acids. These binary sequence patterns are categorized into two classes: Class A patterns match the structural repeat of an idealized amphiphilic α‐helix (3.6 residues per turn), and class B patterns match the structural repeat of an idealized amphiphilic β‐strand (2 residues per turn). The difference between these two classes of sequence patterns has led to a strategy for de novo protein design based on binary patterning of polar and nonpolar amino acids. Here we ask whether similar binary patterning is incorporated in the sequences and structures of natural proteins. Analysis of the Protein Data Bank demonstrates the following. (1) Class A sequence patterns occur considerably more frequently in the sequences of natural proteins than would be expected at random, but class B patterns occur less often than expected. (2) Each pattern is found predominantly in the secondary structure expected from the binary strategy for protein design. Thus, class A patterns are found more frequently in α‐helices than in β‐strands, and class B patterns are found more frequently in β‐strands than in α‐helices. (3) Among the α‐helices of natural proteins, the most commonly used binary patterns are indeed the class A patterns. (4) Among all β‐strands in the database, the most commonly used binary patterns are not the expected class B patterns. (5) However, for solvent‐exposed β‐strands, the correlation is striking: All β‐strands in the database that contain the class B patterns are exposed to solvent. (6) The bias of class A patterns for α‐structure over β‐structure and the bias of class B patterns for β‐structure over α‐structure are significant, not merely when compared to other binary patterns of polar (○) and nonpolar (•) amino acids, but also when compared to the full range of sequences in the database. The implications for the design of novel proteins are discussed.
Posttranslational modifications of bovine osteopontin: Identification of twenty‐eight phosphorylation and three O‐glycosylation sites
Authors: Sørensen Esben S. , Petersen Torben E. , Højrup Peter 
Published: February 2009

AbstractOsteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S‐ethylcysteine‐derivatized phosphopeptides, and mass spectrometric analysis. Twenty‐five phosphoserines and one phosphothreonine were located in Ser/Thr‐X‐Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser‐X‐X‐Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11–32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O‐glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine‐and proline‐rich region of the protein. Three putative N‐glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O‐glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well‐conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.
Design and structural analysis of an engineered thermostable chicken lysozyme
Authors: Shih Phoebe , Kirsch Jack F. 
Published: February 2009

AbstractA hyperstable (hs) variant of chicken egg‐white lysozyme with enhanced thermal (ΔTm ≈︁ +10.5 °C) and chemical (ΔCm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild‐type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 °C) to 4.0 (differential scanning calorimetry, 74 °C) kcal mol−1 greater than that of WT. The specific activity of the hs variant is 2.5‐fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with ΔTm = 3.3 °C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050–2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.
Characterization of the isolated cAMP‐binding B domain of cAMP‐dependent protein kinase
Authors: Shabb John B. , Kapphahn Mark A. , Muhonen Wallace M. , Poteet Celeste E. , Baker Nancy E. , Corbin Jackie D. 
Published: February 2009

AbstractA 14.4‐kDa cAMP‐binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP‐dependent protein kinase type Iα regulatory subunit (RIα). The full‐length RIα from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser‐252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RIα, the [3H]cAMP exchange rate was comparable to that of the B domain of full‐length RIα containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high‐affinity cAMP‐binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2‐mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP‐binding domain can function independently of any other domain structures of the R subunit.