bluuebunny/crossref_metadata_2025 huggingface dataset:
Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding
Authors: Van Ophem Peter W. , Pospischil Maria A. , Manning James M. , Ringe Dagmar , Peisach Daniel , Petsko Gregory , Soda Kenji
Published: February 2009

Of the major amino acid side chains that anchor pyridoxal 5′‐phosphate at the coenzyme binding site of bacterial D‐amino acid transaminase, two have been substituted using site‐directed mutagenesis. Thus, Ser‐180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr‐31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild‐type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5‐15% lower concentration of urea or guanidine than did the wild‐type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5′‐phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild‐type and the S180A enzymes in the presence of normal D‐amino acid substrates. However, β‐chloro‐D‐alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild‐type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5‐phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild‐type enzymes because racemase activity, i.e., conversion of L‐alanine to D‐alanine, was higher than for the wild‐type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.
Nuclear magnetic resonance characterization of the N‐terminal thioredoxin‐like domain of protein disulfide isomerase
Authors: Kemmink Johan , Darby Nigel J. , Creighton Thomas E. , Dijkstra Klaas , Scheek Ruud M.
Published: February 2009

A genetically engineered protein consisting of the 120 residues at the N‐terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and β‐sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N‐terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains.
Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions
Authors: Delboni Luis F. , Mande Shekhar C. , Turley Stewart , Hol WIM G.J. , Rentier‐Delrue Françoise , Mainfroid Véronique , Martial Joseph A. , Vellieux Frederique M.D.
Published: February 2009

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2‐phosphoglycolate was determined by X‐ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the “closed” conformation in either subunit. The crystallographic R‐factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 Å for 248 Cα atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual ϕ,ψ combination.

A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.
Recombinant protein sequences can trigger methylation of N‐terminal amino acids in Escherichia coli
Authors: Apostol Izydor , Aitken Jackie , Levine Joe , Lippincott Julie , Davidson Jeffrey S. , Abbott‐Brown Debbie
Published: February 2009

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild‐type valine residues at all N‐termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N‐terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N‐terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N‐terminal alanine that occurred through two posttrans‐lational events: initial excision of the N‐terminal methionine, followed by methylation of alanine as the newly generated N‐terminus. No methylation was observed for variants expressed with wild‐type valine at the N‐terminus of the alpha globin. The methylation of N‐terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N‐terminal methylation has been observed previously for native E. coli proteins with similar N‐terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.
Protein recognition of ammonium cations using side‐chain aromatics: A structural variation for secondary ammonium ligands
Authors: Raine Andrew R.C. , Yang Chien‐Chih , Packman Leonard C. , White Scott A. , Mathews F. Scott , Scrutton Nigel S.
Published: February 2009

A model for the structure of dimethylamine dehydrogenase was generated using the crystal coordinates of trimethylamine dehydrogenase. Substrate is bound in trimethylamine dehydrogenase by cation‐7r bonding, but modeling of dimethylamine dehydrogenase suggests that secondary amines are bound by a mixture of cation‐7r and conventional hydrogen bonding. In dimethylamine dehydrogenase, binding is orientationally more specific and distinct from those proteins that bind tertiary and quaternary amine groups.
Energetics of hydrogen bonding in proteins: A model compound study
Authors: Habermann Susan M. , Murphy Kenneth P.
Published: February 2009

Differences in the energetics of amide‐amide and amide‐hydroxyl hydrogen bonds in proteins have been explored from the effect of hydroxyl groups on the structure and dissolution energetics of a series of crystalline cyclic dipeptides. The calorimetrically determined energetics are interpreted in light of the crystal structures of the studied compounds. Our results indicate that the amide‐amide and amide‐hydroxyl hydrogen bonds both provide considerable enthalpic stability, but that the amide‐amide hydrogen bond is about twice that of the amide‐hydroxyl. Additionally, the interaction of the hydroxyl group with water is seen most readily in its contributions to entropy and heat capacity changes. Surprisingly, the hydroxyl group shows weakly hydrophobic behavior in terms of these contributions. These results can be used to understand the effects of mutations on the stability of globular proteins.
Multiple domain protein diagnostic patterns
Authors: Adams R. Mark , Das Sudeshna , Smith Temple F.
Published: February 2009

We have implemented an iterative algorithm for the identification of diagnostic patterns from sets of multiple‐domain proteins, where domains need not be common to all the proteins in the defining set. Our algorithm was applied to sequences gathered using a variety of methods, including BLAST, common keywords, and common E.C. numbers. In all cases, useful diagnostic patterns were obtained, possessing both high sensitivity and specificity. The patterns were found to correlate in several cases with both functional and structural domains. Patterns generated from a large number of sequence families were analyzed for probable multiple‐domain structure.
The crystal structure of TGF‐β3 and comparison to TGF‐β2: Implications for receptor binding
Authors: Mittl Peer R. E. , Priestle John P. , Cox David A. , Mcmaster Gary , Cerletti Nico , Grütter Markus G.
Published: February 2009

Transforming growth factors β belong to a group of cytokines that control cellular proliferation and differentiation. Five isoforms are known that share approximately 75% sequence identity, but exert different biological activities. The structure of TGF‐β3 was solved by X‐ray crystallography and refined to a final R‐factor of 17.5% at 2.0 Å resolution. Comparison with the structure of TGF‐β2 (Schlunegger MP, Grütter MG, 1992, Nature 358:430‐434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 257:369‐373) reveals a virtually identical central core. Differences exist in the conformations of the N‐terminal α‐helix and in the β‐sheet loops. In TGF‐β3, the N‐terminal α‐helix has moved ≈︁ 1 Å away from the central core. This movement can be correlated with the mutation of Leu 17 to Val and Ala 47 to Pro in TGF‐β3. The β‐sheet loops rotate as a rigid body 9° around an axis that runs approximately parallel to the dimer axis. If these differences are recognized by the TGF‐β receptors, they might account for the individual cellular responses. A molecule of the precipitating agent dioxane is bound in a crystal contact, forming a hydrogen bond with Trp 32. This dioxane may occupy a carbohydrate‐binding site, because dioxane possesses some structural similarity with a carbohydrate. The dioxane is in contact with two tryptophans, which are often involved in carbohydrate recognition.
Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit
Authors: Grant Bruce D. , Tsigelny Igor , Taylor Susan S. , Adams Joseph A.
Published: February 2009

The active site of the cAMP‐dependent protein kinase catalytic subunit harbors a cluster of acidic residues —Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241 — that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gin (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild‐type enzyme, and displays wild‐type‐like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre‐steady‐state quench‐flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.
Cytochrome c3 from Desulfovibrio gigas: Crystal structure at 1.8 Å resolution and evidence for a specific calcium‐binding site
Authors: Matias Pedro M. , Morais José , Coelho Ricardo , Carrondo Maria Arménia , Wilson Keith , Dauter Zbigniew , Sieker Larry
Published: February 2009

Crystals of the tetraheme cytochrome c3 from sulfate‐reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X‐ray diffraction data collected to 1.8 Å resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R‐factor of 14.9%. The three‐dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N‐terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme‐redox perturbation, and might even be involved in the specificity of recognition with its redox partner.
Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM‐3 using site‐directed mutagenesis
Authors: Govindaraj Shanthi , Poulos Thomas L.
Published: February 2009

Cytochrome P450BM‐3 is a catalytically self‐sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 54:11221‐11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin‐to‐heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six‐proline substitution, did not change significantly relative to wild‐type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin‐to‐heme electron transfer.
The protein phosphatase 2C (PP2C) superfamily: Detection of bacterial homologues
Authors: Bork Peer , Brown Nigel P. , Hegyi Hedvig , Schultz Jörg
Published: February 2009

A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.
Crystallization and preliminary X‐ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus
Authors: Moser Markus , Müller Rita , Jansonius Johan N. , Battchikova Natalia , Koivulehto Marianne , Korpela Timo
Published: February 2009

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X‐rays to at least 2.0 Å nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 Å, b = 93.1 Å, c = 45.6Å, α = 90.0°, β = 106.8°, γ = 90.0°. A native data set to 2.3 Å has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.
X‐ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from rhodobacter capsulatus: Implications for ion selectivity and single‐channel conductance
Authors: Przybylski Michael , Glocker Michael O. , Nestel Uwe , Schnaible Volker , Blüggel Martin , Diederichs Kay , Weckesser Jurgen , Schad Michael , Schmid Angela , Welte Wolfram , Benz Roland
Published: February 2009

The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinyl‐porins determined by mass spectrometry and X‐ray crystallography. Molecular weight and peptide mapping analyses using matrix‐assisted laser desorption‐ionization mass spectrometry identified selective succinylation of three lysine‐ϵ‐amino groups (Lys‐46, Lys‐298, Lys‐300) and the N‐terminal α‐amino group. The structure of a tetra‐succinylated porin (TS‐porin) was determined to 2.4 Å and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di‐succinylated porin (MS‐porin) and a TS‐porin are localized at the inner channel surface and are solvent‐accessible: Lys‐46 is located at the channel constriction site, whereas Lys‐298, Lys‐300, and the N‐terminus are all near the periplasmic entrance of the channel. The Lys‐46 residue at the central constriction loop was modeled as succinyl‐lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS‐ and TS‐porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single‐channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure‐selective modification of charged residues as an efficient approach in the structure‐function evaluation of ion channels, and X‐ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.
The (QxW)3 domain: A flexible lectin scaffold
Authors: Hazes Bart
Published: February 2009

Lectins form a class of proteins that have evolved a specialized carbohydrate‐binding function. Based on amino acid sequence analysis, several lectin families have been described and a lectin domain, the (QxW)3 domain, was discussed recently based on 11 family members. In this paper, the (QxW)3 domain family is extended to 45 sequences, several of which have very low sequence identity with the previously known members of the family. A hidden Markov model was used to identify the most divergent members of the family. The expanded set of sequences gives us a more complete appreciation of the conserved features, and the lack thereof, in this lectin family. This, in turn, provides new insights in the structural and functional properties of the individual family members.
A novel complex of a phenolic derivative with insulin: Structural features related to the T → R transition
Authors: Smith G.David , Ciszak Ewa , Pangborn Walter
Published: February 2009

The structure of a symmetric T3Rf3 insulin hexamer, complexed with 4‐hydroxybenzamide, has been determined using X‐ray crystallographic techniques. Data were measured from six crystals grown in microgravity to a resolution of 1.4 Å and the structure has been refined including the contributions from hydrogen atoms. The crystals are isomorphous with T3Rf3 complexes of phenolic derivatives as well as with uncomplexed forms. Unlike the structures of complexes with phenol, m‐cresol, resorcinol, 4′‐hydroxyacetanilide, and methylparaben, which bind one phenolic derivative molecule per R‐ or Rf‐state monomer, two molecules of 4‐hydroxybenzamide are bound by each Rf‐state monomer. The presence of the second guest molecule results in an extensive hydrogen bonding network, mediated by water molecules, between the T‐ and Rf‐state trimers and adds stability to the formation of the hexamer. The only access to these second sites is through three symmetry‐related, narrow channels that originate on the surface of the T‐state trimer. Although the conformation of the backbone atoms of the monomers is nearly identical to that of other T3Rf3 hexamers, significant changes are observed in the conformations of side chains in the vicinity of the second binding site. The side chain of the T‐state A11 Cys residue, which forms a disulfide bond to A6 Cys in the same monomer, is observed in two discrete conformations; two discrete conformations are also present for the entire A8 Thr residue in the Rf‐state monomer. A procedure is also described for an alternate method of interframe scaling and merging intensity data from an image plate detector.
The NMR solution structure of the pheromone Er‐11 from the ciliated protozoan Euplotes raikovi
Authors: Luginbühl Peter , Wu Jihui , Zerbe Oliver , Ortenzi Claudio , Luporini Pierangelo , Wüthrich Kurt
Published: February 2009

The NMR solution structure of the pheromone Er‐11, a 39‐residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, Cα, and C of residues 2–38 was 0.30 Å. The molecular architecture is dominated by an up‐down‐up bundle of three short helices with residues 2–9, 12–19, and 22–32, which is closely similar to the previously determined structures of the homologous pheromones EM, Er‐2, and EMO. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell‐surface receptors.
Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy
Authors: Freskgård Per‐Ola , Olsen Ole H. , Persson Egon
Published: February 2009

Factor VIIa (fVIIa) is composed of four discrete domains, a 7‐carboxyglutamic acid (Gla)‐containing domain, two epidermal growth factor (EGF)‐like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF‐like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca2+‐dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca2+‐induced changes in the far‐UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca2+‐induced ordering of α‐helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa constructed on the basis of known structures of homologous proteins. Far‐UV spectra of the Gla domain of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K‐dependent plasma proteins. The near‐UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.
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

Here 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

Because 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

Utilizing 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

The 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

The 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

Although 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

For 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

An 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

The 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

The 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

We 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

Peptides 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

Adrenodoxin 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

In 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.