bluuebunny/crossref_metadata_2025_split huggingface dataset:
What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties
Authors: Munson Mary , Balasubramanian Suganthi , Fleming Karen G. , Nagi Athena D. , O'Brien Ronan , Sturtevant Julian M. , Regan Lynne 
Published: February 2009

AbstractHere we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four‐helix‐bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native‐like properties of the wild‐type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with larger side chains causes a loss of native‐like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native‐like proteins.
Ribosome‐mediated translational pause and protein domain organization
Authors: Thanaraj T.A. , Argos Patrick 
Published: February 2009

AbstractBecause regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain. Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three‐dimensional structures. The frequencies of individual codons in mRNAs of highly expressed genes from E. coli were taken as a measure of codon translation speed. Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions. The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed. The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined. The results indicated that protein domain boundaries that mark higher‐order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm. The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome. The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding. Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis.
β‐Methylthio‐aspartic acid: Identification of a novel posttranslational modification in ribosomal protein S12 from escherichia coli
Authors: Kowalak Jeffrey A. , Walsh Kenneth A. 
Published: February 2009

AbstractUtilizing microscale chemical derivatization reactions and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, β‐methylthio‐aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome.
Three‐dimensional structure of the complexes of ribonuclease A with 2′,5′‐CpA and 3′,5′‐d(CpA) in aqueous solution, as obtained by NMR and restrained molecular dynamics
Authors: Toiron Catherine , Gonzalez Carlos , Bruix Marta , Rico Manuel 
Published: February 2009

AbstractThe three‐dimensional structure of the complexes of ribonuclease A with cytidyl‐2′,5′‐adenosine (2′,5′‐CpA) and deoxycytidyl‐3′,5′‐deoxyadenosine [3′,5′‐d(CpA)] in aqueous solution has been determined by 1H NMR methods in combination with restrained molecular dynamics calculations. Twenty‐three intermolecular NOE cross‐correlations for the 3′,5′‐d(CpA) complex and 19 for the 2′,5′‐CpA, together with about 1,000 intramolecular NOEs assigned for each complex, were translated into distance constraints and used in the calculation. No significant changes in the global structure of the enzyme occur upon complex formation. The side chains of His 12, Thr 45, His 119, and the amide backbone group of Phe 120 are involved directly in the binding of the ligands at the active site. The conformation of the two bases is anti in the two complexes, but differs from the crystal structure in the conformation of the two sugar rings in 3′,5′‐d(CpA), shown to be in the S‐type region, as deduced from an analysis of couplings between the ribose protons. His 119 is found in the two complexes in only one conformation, corresponding to position A in the free protein. Side chains of Asn 67, Gln 69, Asn 71, and Glu 111 form transient hydrogen bonds with the adenine base, showing the existence of a pronounced flexibility of these enzyme side chains at the binding site of the downstream adenine. All other general features on the structures coincide clearly with those observed in the crystal state.
Role of the divalent metal ion in the NAD:malic enzyme reaction: An ESEEM determination of the ground state conformation of malate in the E:Mn: Malate complex
Authors: Tipton Peter A. , Quinn Thomas P. , Peisach Jack , Cook Paul F. 
Published: February 2009

AbstractThe conformation of L‐malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2‐position, the 3R‐position, and the 3S‐position of L‐malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn‐malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L‐malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L‐malate at the active site of the enzyme.
Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments
Authors: Gordon Larry M. , Horvath Suzanna , Longo Marjorie L. , Zasadzinski Joseph A.N. , Taeusch H.William , Faull Kym , Leung Carol , Waring Alan J. 
Published: February 2009

AbstractAlthough the effects of surfactant protein B (SP‐B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure‐activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N‐terminal SP‐B peptides in lipids and structure‐promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP‐B1–25 (residues 1–25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP‐B1–25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP‐B1–25 sequence. Residues Cys‐8‐Gly‐25 are largely α‐helix in methanol, whereas the N‐terminal segment Phe‐1‐Cys‐8 had turn and helical propensities. Addition of SP‐B1–25 spin‐labeled at the N‐terminal Phe (i.e., SP‐B*1–25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP‐B1–25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP‐B1–25 argues that the reporter on Phe‐1 resides in the headgroup of the lipid dispersions. The blue‐shift in the endogenous fluorescence of Trp‐9 near the N‐terminus of SP‐B1–25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP‐B1–25 interacting with lipids.
Hydrophobic regions on protein surfaces. Derivation of the solvation energy from their area distribution in crystallographic protein structures
Authors: Eisenhaber Frank 
Published: February 2009

AbstractFor the first time, a direct approach for the derivation of an atomic solvation parameter from macromolecular structural data alone is presented. The specific free energy of solvation for hydrophobic surface regions of proteins is delineated from the area distribution of hydrophobic surface patches. The resulting value is 18 cal/(mol‐Å2), with a statistical uncertainty of ±2 cal/(mol‐Å2) at the 5% significance level. It compares favorably with the parameters for carbon obtained by other authors who use the the crystal geometry of succinic acid or energies of transfer from hydrophobic solvent to water for small organic compounds. Thus, the transferability of atomic solvation parameters for hydrophobic atoms to macromolecules has been directly demonstrated.A careful statistical analysis demonstrates that surface energy parameters derived from thermodynamic data of protein mutation experiments are clearly less confident.
Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability
Authors: Eberhardt Eric S. , Wittmayer Paula K. , Templer Barbra M. , Raines Ronald T. 
Published: February 2009

AbstractAn intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent‐inaccessible side chain that donates a hydrogen bond to the main‐chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate‐limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr π Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41‐Pro 42 peptide bond.
Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in mycoplasma capricolum
Authors: Zhu Peng‐Peng , Peterkofsky Alan 
Published: February 2009

AbstractThe region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)‐lplA (encoding lipoate‐protein ligase)‐odpA (encoding pyruvate dehydrogenase EIα)‐odpB (encoding pyruvate dehydrogenase EIβ)‐odp2 (encoding pyruvate dehydrogenase EII)‐dldH (encoding dihydrolipoamide dehydrogenase)‐pta (encoding phosphotransacetylase)‐ack: (encoding acetate kinase)‐orfA (an unknown open reading frame)‐kdtB‐ptsI‐crr. Analysis of the DNA sequence suggests that the naox and IplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram‐positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M. genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino‐terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.
Electrostatic effects in electron transfer reactions of [2Fe‐2S] ferredoxins with inorganic reagents
Authors: Vidakovic Momcilo , Germanas Juris P. 
Published: February 2009

AbstractThe kinetics of electron transfer from the reduced [2Fe‐2S] ferredoxins from the cyanobacterium Anabaena 7120 and the protozoan Trichomonas vaginalis to select cobalt coordination compounds have been studied in order to gain insight into the mechanism of electron transfer and intrinsic reactivity of [2Fe‐2S] active sites. With tripositive cobalt complexes, reactions of both proteins displayed saturation kinetics; values of association constants of 12,900 and 1,400 M−1 and limiting rate constants of 7.6 and 3.5 s−1 were found for oxidation of T. vaginalis and Anabaena ferredoxins, respectively, by Co(NH3)63+ at room temperature and I = 0.1 M. An activation enthalpy of 12.1 kcal/mol and activation entropy of — 14.3 cal/mol K for oxidation of T. vaginalis ferredoxin by Co(NH3)63+ contrasted with corresponding values of 13.4 kcal/mol and — 10.5 cal/mol K for the Spirulina platensis protein, which is homologous to Anabaena ferredoxin. The dependence of the reaction rates on ionic strength were measured to probe the importance of electrostatics on the reactivity of the proteins. Analysis of the ionic strength dependence of the oxidation of the proteins by Co(NH3)63+ by the “parallel plate” model of Watkins et al. (1994, Protein Sci 3:2104–2114) afforded values for active site charges of — 0.7 and — 1.1 and limiting rate constants at infinite ionic strength of 25,800 and 76 M−1s−1 for T. vaginalis and Anabaena ferredoxins, respectively. These results suggest that the [2Fe‐2S] center of the protozoal ferredoxin is more accessible and adjacent to a less highly charged, more compact patch of negative charges than the photosynthetic protein.
Improved genetic algorithm for the protein folding problem by use of a Cartesian combination operator
Authors: Rabow Alfred A. , Scheraga Harold A. 
Published: February 2009

AbstractWe have devised a Cartesian combination operator and coding scheme for improving the performance of genetic algorithms applied to the protein folding problem. The genetic coding consists of the Cα Cartesian coordinates of the protein chain. The recombination of the genes of the parents is accomplished by: (1) a rigid superposition of one parent chain on the other, to make the relation of Cartesian coordinates meaningful, then, (2) the chains of the children are formed through a linear combination of the coordinates of their parents. The children produced with this Cartesian combination operator scheme have similar topology and retain the long‐range contacts of their parents. The new scheme is significantly more efficient than the standard genetic algorithm methods for locating low‐energy conformations of proteins. The considerable superiority of genetic algorithms over Monte Carlo optimization methods is also demonstrated. We have also devised a new dynamic programming lattice fitting procedure for use with the Cartesian combination operator method. The procedure finds excellent fits of real‐space chains to the lattice while satisfying bond‐length, bond‐angle, and overlap constraints.
Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4
Authors: Ren Z.J. , Black L.W. , Lewis G.K. , Wingfield P.T. , Locke E.G. , Steven A.C. 
Published: February 2009

AbstractPeptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self‐assemble. We describe here a system in which the molecules to be displayed are bound to pre‐assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc‐deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C‐terminal fusions to SOC of (1) a tetrapeptide; (2) the 43‐residue V3 loop domain of gpl20, the human immunodeficiency virus type‐1 (HIV‐1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC‐V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to ∼ 103 copies per capsid and >104 copies per polyhead of V3‐sized domains. Phage displaying SOC‐VP1 were isolated from a 1:106 mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.
Conformational stability of adrenodoxin mutant proteins
Authors: Burova Tatjana V. , Beckert Vita , Ristau Otto , Uhlmann Heike , Bernhardt Rita , Pfeil Wolfgang 
Published: February 2009

AbstractAdrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4–114 and 4–108, were studied by high‐sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 °C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change δCp = 7.28 ± 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (ΔCp = 4.29 ± 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4–108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.
A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease
Authors: Wang Yi , Shortle David 
Published: February 2009

AbstractIn an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortle D, 1995, Biochemistry 34:1589515905), we reported evidence of a three‐strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local “non‐native” interaction with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, this unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data that confirm the important role of interactions involving four hydrophobic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the structure formed by the chain segments corresponding to β1‐β2‐β3‐h, interactions that are not present in the native state. Glycine substitutions for each of these large hydrophobic residues destabilizes or disrupts this beta structure, as assessed by HN line sharpening and changes in the CD spectrum. The 13C resonances of the carbonyl carbon for several of the residues in this structure indicate conformational dynamics that respond in a complex way to addition of urea or changes in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native‐like three‐strand beta sheet. Instead, the data are more consistent with the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by hydrophobic interactions.