bluuebunny/crossref_metadata_2025 huggingface dataset:
The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells
Authors: Papov Vladimir V. , Gravina Stephen A. , Mieyal John J. , Biemann Klaus
Published: July 2010

Thioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088–6097). The primary sequence of the HPLC‐pure enzyme was determined by tandem mass spectrometry and found to represent a 105‐amino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins called glutaredoxins among the family of thiol: disulfide oxidoreductases that also includes thioredoxin and protein disulfide isomerase. Although this human red blood Cell glutaredoxin (hRBC Grx) is highly homologous to the 3 other mammalian Grx proteins whose sequences are known (calf thymus, rabbit bone marrow, and pig liver), there are a number of significant differences. Most notably an additional cysteine residue (Cys‐7) occurs near the N‐terminus of the human enzyme in place of a serine residue in the other proteins. In addition, residue 51 of hRBC Grx displayed a mixture of Asp and Asn. This result is consistent with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the enzyme was prepared from blood combined from a number of individual donors, it is not clear whether this Asp/Asn ambiguity represents inter‐individual variation, gene duplication, or a deamidation artifact of purification.
Novel phosphotransferase system genes revealed by bacterial genome analysis: Unique, putative fructose‐ and glucoside‐specific systems
Authors: Reizer Jonathan , Michotey Valerie , Reizer Aiala , Saier Milton H.
Published: July 2010

Analyses of sequences made available through the Escherichia coli genome project in the 87.2‐89.2‐min and 81.5‐84.5‐min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate: sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582. ORFf147 and ORFf485 comprise an Enzyme IIA‐Enzyme IIBC pair of the PTS. The sequence similarity of ORFf485 to previously characterized fructose‐specific Enzymes IIBC suggests that ORFf485 may be specific for fructose. ORFf 147 encodes a protein with comparable degrees of sequence similarity to fructose and mannitol‐specific Enzymes IIA as well as homologous proteins implicated in σ54‐dependent transcriptional regulation. Unique features of this system include a detached IIA protein and the absence of a IIB domain duplication. ORFf356 and ORFf582 are functionally unidentified and nonhomologous to other ORFs in the current protein databases, but ORFf582 contains 2 N‐terminal helix‐turn‐helix motifs, suggestive of a role in frv operon transcriptional regulation.

The second putative operon, designated glv, includes 3 ORFs, ORFf455, ORFf 161, and ORFf212. We suggest that ORFf455 was incorrectly assigned and should be designated ORFf368. ORFf368 and ORFfl61 encode an Enzyme IIC and IIB pair of the PTS showing greatest sequence similarity to Enzymes II specific for sugars of the gluco configuration. ORFf212 encodes a protein with sequence similarity to a phospho‐β‐glucosidase and an α‐galactosidase. No putative transcriptional regulator of the glv operon was found. This operon is the first one encoding a putative PTS permease with detached Enzymes IIB and IIC and lacking an Enzyme IIA. It is suggested that both the frv and glv operons are cryptic in E. coli and that additional genes encoding novel PTS‐related proteins will be revealed by bacterial genome sequence analyses.
Multiple copy sampling in protein loop modeling: Computational efficiency and sensitivity to dihedral angle perturbations
Authors: Zheng Qiang , Rosenfeld Rakefet , Delisi Charles , Kyle Donald J.
Published: July 2010

Multiple copy sampling and the bond scaling‐relaxation technique are combined to generate 3‐dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20‐50% of the computational time required by the single‐copy method for the various protein segments tested. An analytical formula is proposed to estimate the computational gain prior to carrying out a multicopy simulation. When 7‐residue loops within flexible proteins are modeled, each multicopy simulation can sample a set of loop conformations with initial dispersions up to ±15° for backbone and ±3° for side‐chain rotatable dihedral angles. The dispersions are larger for shorter and smaller for longer and/or surface loops. The degree of convergence of loop copies during a simulation can be used to complement commonly used target functions (such as potential energy) for distinguishing between native and misfolded conformations. Furthermore, this convergence also reflects the conformational flexibility of the modeled protein segment. Application to simultaneously building all 6 hypervariable loops of an antibody is discussed.
Protein folding dynamics: The diffusion‐collision model and experimental data
Authors: Karplus Martin , Weaver David L.
Published: July 2010

The diffusion‐collision model of protein folding is assessed. A description is given of the qualitative aspects and quantitative results of the diffusion‐collision model and their relation to available experimental data. We consider alternative mechanisms for folding and point out their relationship to the diffusion‐collision model. We show that the diffusion‐collision model is supported by a growing body of experimental and theoretical evidence, and we outline future directions for developing the model and its applications.
Determination of the binding frame within a physiological ligand for the chaperone SecB
Authors: Topping Traci B. , Randall Linda L.
Published: July 2010

The hallmark of the class of proteins called chaperones is the amazing ability to bind tightly to a wide array of polypeptide ligands that have no consensus in sequence; chaperones recognize non‐native structure. As a step in the elucidation of the molecular mechanism of such remarkable binding, we have characterized complexes between the bacterial chaperone SecB and a series of ligands related to maltose‐binding protein. SecB interacts at multiple sites on its polypeptide ligand. The entire binding region covers approximately half of the primary sequence of maltose‐binding protein and comprises contiguous sites positioned around the center of the sequence.
Modeling studies of the change in conformation required for cleavage of limited proteolytic sites
Authors: Hubbard S.J. , Eisenmenger F , Thornton J.M.
Published: July 2010

Previous analyses of limited proteolytic sites within native, folded protein structures have shown that a significant conformational change is required in order to facilitate binding into the active site of the attacking proteinase. For the serine proteinases, the optimum conformation to match the proteinase binding‐site geometry has been well characterized crystallographically by the conserved main‐chain geometry of the reactive site loops of their protein inhibitors. A good substrate must adopt a conformation very similar to this “target” main‐chain conformation prior to cleavage. Using a “loop‐closure” modeling approach, we have tested the ability of a set of trypticlimited proteolytic sites to achieve this target conformation and further tested their suitability for cleavage. The results show that in most cases, significant changes in the conformation of at least 12 residues are required. All the putative tryptic cleavage sites in 1 protein, elastase, were also modeled and tested to compare the results to the actual nicksite in that protein. These results strongly suggest that large local motions proximate to the scissile bond are required for proteolysis, and it is this ability to unfold locally without perturbing the overall protein conformation that is the prime determinant for limited proteolysis.
Three‐dimensional, sequence order‐independent structural comparison of a serine protease against the crystallographic database reveals active site similarities: Potential implications to evolution and to protein folding
Authors: Fischer Daniel , Wolfson Haim , Lin Shuo L. , Nussinov Ruth
Published: July 2010

We have recently developed a fast approach to comparisons of 3‐dimensional structures. Our method is unique, treating protein structures as collections of unconnected points (atoms) in space. It is completely independent of the amino acid sequence order. It is unconstrained by insertions, deletions, and chain directionality. It matches single, isolated amino acids between 2 different structures strictly by their spatial positioning regardless of their relativesequentialposition in the amino acid chain. It automatically detects a recurring 3D motif in protein molecules. No predefinition of the motif is required. The motif can be either in the interior of the proteins or on their surfaces. In this work, we describe an enhancement over our previously developed technique, which considerably reduces the complexity of the algorithm. This results in an extremely fast technique. A typical pairwise comparison of 2 protein molecules requires less than 3 s on a workstation. We have scanned the structural database with dozens of probes, successfully detecting structures that are similar to the probe. To illustrate the power of this method, we compare the structure of a trypsin‐like serine protease against the structural database. Besides detecting homologous trypsin‐like proteases, we automatically obtain 3D, sequence order‐independent, active‐site similarities with subtilisin‐like and sulfhydryl proteases. These similarities equivalence isolated residues, not conserving the linear order of the amino acids in the chains. The active‐site similarities are well known and have been detected by manually inspecting the structures in a time‐consuming, laborious procedure. This is the first time such equivalences are obtained automatically from the comparison of full structures. The far‐reaching advantages and the implications of our novel algorithm to studies of protein folding, to evolution, and to searches for pharmacophoric patterns are discussed.
A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the α/β hydrolase fold, acts as a pH‐dependent protein‐stabilizing element
Authors: Mortensen Uffe H. , Breddam Klaus
Published: July 2010

Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low‐barrier type, with another acidic residue, hence forming a “glutamic acid bridge.” In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site‐specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.
Association of the catalytic subunit of aspartate transcarbamoylase with a zinc‐containing polypeptide fragment of the regulatory chain leads to increases in thermal stability
Authors: Peterson Cynthia B. , Zhou Bin‐Bing , Hsieh Durwynne , Creager Angela N.H. , Schachman H. K.
Published: July 2010

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70‐amino acid zinc‐containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 °C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 °C. The values of tm for a variety of complexes of mutant C trimers and the wild‐type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation. In an extreme case with a mutant form involving replacement of Glu 86 by Ala in the catalytic chains, this was manifested by a change in tm for the trimer of 44.6 °C to 64.6 °C for the holoenzyme. These results contribute to our understanding of an earlier observation that scanning calorimetry on wild‐type ATCase gave 2 transitions, with the high temperature peak, which is assigned to melting of C trimers, exhibiting a higher tm than isolated C trimer. The effect of the zinc domain on the tm of the complex with C trimer provides an explanation for this increase in thermal stability, i.e., during heat denaturation of the holoenzyme, the C trimer is still associated with the folded zinc domain fragments of regulatory chains.
High‐sensitivity determination of tyrosine‐phosphorylated peptides by on‐line enzyme reactor and electrospray ionization mass spectrometry
Authors: Amankwa Lawrence N. , Harder Kenneth , Jirik Frank , Aebersold Ruedi
Published: July 2010

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine‐phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on‐line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments.

The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase β onto the inner surface of a small piece of a 50‐μm inner diameter, 360‐μm outer diameter fused silica capillary or by immobilization of the phosphatase onto 40–90‐μm avidin‐activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on‐line electrospray ionization mass spectrometer

Tyrosine‐phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase‐induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides

We demonstrate that, with this technique, low picomole amounts of a tyrosine‐phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.
Two domains of superfamily I helicases may exist as separate proteins
Authors: Koonin Eugene V. , Rudd Kenneth E.
Published: July 2010

DNA and RNA helicases of superfamily I are characterized by seven conserved motifs. The five N‐terminal motifs are separated from the two C‐terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains. Using computer methods for protein sequence analysis, we show that PhoH, an ATP‐binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N‐terminal domain of the helicases, whereas the putative E. coli protein YjhR is homologous to the C‐terminal domain. These findings suggest that the N‐ and C‐terminal domains of superfamily I helicases have distinct activities, with only the N‐terminal domain having the ATPase activity. It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C‐ and N‐terminal domain, respectively.
PROMOTIF—A program to identify and analyze structural motifs in proteins
Authors: Hutchinson E. Gail , Thornton Janet M.
Published: July 2010

We describe a suite of programs, PROMOTIF, that analyzes a protein coordinate file and provides details about the structural motifs in the protein. The program currently analyzes the following structural features: secondary structure; β‐ and γ‐turns; helical geometry and interactions; β‐strands and β‐sheet topology; β‐bulges; β‐hairpins; β‐α‐β units and Ψ‐loops; disulphide bridges; and main‐chain hydrogen bonding patterns. PROMOTIF creates postscript files showing the examples of each type of motif in the protein, and a summary page. The program can also be used to compare motifs in a group of related structures, such as an ensemble of NMR structures.
Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies
Authors: Schliebs Wolfgang , Thanki Narmada , Eritja Ramon , Wierenga Rik
Published: July 2010

MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild‐type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site‐directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM‐K13A is completely inactive, although it can still bind substrate analogues, and monoTIM‐H95A is 50 times less active. The best inhibitors of wild‐type TIM are phosphoglycolohydroxamate (PGH) and 2‐phosphoglycolate (2PG), with K, values of 8 μM and 26 μM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60‐fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild‐type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second sub‐unit, monoTIM catalyzes the interconversion of D‐glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.
Chemical crosslinking of the subunits of HIV‐1 reverse transcriptase
Authors: Debyser Zeger , De Clercq Erik
Published: July 2010

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV‐1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV‐1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weights greater than that of the dimeric form accumulate during a 1–15‐min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher‐order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV‐1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV‐1 RT.
Determinants of performance in the isocitrate dehydrogenase of Escherichia coli
Authors: Dean Antony M. , Shiau Andrew K. , Koshland Daniel E.
Published: July 2010

The substrate specificity of the NADP‐dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site‐directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild‐type enzyme with 2R‐malate reveals that the γ‐carboxylate of 2R, 3S‐isocitrate contributes a factor of 12, 000, 000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1, 650 can be ascribed to the hydrogen bond formed between S113 and the γ‐carboxylate of bound isocitrate, a factor of 150 to the negative charge of the γ‐carboxylate, and a factor of 50 for the γ‐methyl. These results are entirely consistent with X‐ray structures of Michaelis complexes that show a hydrogen bond positions the γ‐carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.
Synthesis, structure, and structure‐activity relationships of divalent thrombin inhibitors containing an α‐keto‐amide transition‐state mimetic
Authors: Krishnan Raman , Tulinsky A. , Vlasuk George P. , Pearson Daniel , Vallar Pureza , Bergum Peter , Brunck Terence K. , Ripka William C.
Published: July 2010

A new class of divalent thrombin inhibitors is described that contains an α‐keto‐amide transition‐state mimetic linking an active site binding group and a group that binds to the fibrinogen‐binding exosite. The X‐ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition‐state‐like binding of the α‐keto‐amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side‐chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition‐state‐containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their α‐keto‐amide counterparts. The most potent cv‐keto‐amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.
Two crystal structures of the leupeptin‐trypsin complex
Authors: Kurinov I. V. , Harrison R. W.
Published: July 2010

Three‐dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 Å resolution with R‐factor = 17.7% in the trigonal crystal space group P3, 21, with unit cell dimensions of a = b = 55.62 Å, c = 110.51 Å. The second structure was determined at a resolution of 1.8 Å with R‐factor = 17.5% in the orthorhombic space group P212121, with unit cell dimensions of a = 63.69 Å, b = 69.37 Å, c = 63.01 Å. The overall protein structure is very similar in both crystal forms, with RMS difference for main‐chain atoms of 0.27 Å. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 Å length with side‐chain oxygen of Ser‐195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His‐57.
Crystallization and preliminary structural analysis of Bacillus subtilis adenylosuccinate lyase, an enzyme implicated in infantile autism
Authors: Redinbo Matthew R. , Eide Sithumini M. , Stone Randy L. , Dixon Jack E. , Yeates Todd O.
Published: July 2010

Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X‐ray diffraction is in progress. ASL is a 200‐kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the β‐elimination of fumarate. A single point mutation in the human ASL gene has been linked to mental retardation with autistic features. In addition, ASL plays an important role in the bioprocessing of anti‐HIV therapeutics. B. subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K. The space group is P6122 or P6522 with a = b = 129.4 Å; the length of the c‐axis varies between 275 and 290 Å, depending on the crystal. An analysis of solvent content indicates a dimer in the asymmetric unit, although a self‐rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes. Structure determination by isomorphous replacement is in progress.
The structural homology between uteroglobin and the pore‐forming domain of colicin A suggests a possible mechanism of action for uteroglobin
Authors: Cruz Xavier De La , Lee B.
Published: July 2010

Although the exact physiological function of uteroglobin is not known, it has been suggested that it may function by inhibiting phospholipase A2. We have found that the uteroglobin fold is embedded in that of the pore‐forming domain of colicin A. Colicin A is an antibiotic protein that kills sensitive Escherichia coli cells by forming a pore in their phospholipid membrane. The RMS deviation between the Cα atoms after the structural alignment is 2.39 Å for the 52 superimposed residues. In the alignment, uteroglobin helices 1, 2, 3, and 4 align with colicin A helices 6, 7, 3, and 4, respectively. The motif is strongly amphipathic in both proteins. On the basis of this common structural motif and of known experimental data on both proteins, we propose that UG binds to the membrane surface by lying on it monotopically. The phospholipase A2 inhibition would follow this initial binding step.
Energetics of structural domains in α‐lactalbumin
Authors: Hendrix Tonya M. , Griko Yuri , Privalov Peter
Published: July 2010

α‐Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in α‐helix (the α‐domain) and has Cys 6‐Cys 120 and Cys 28‐Cys 111 disulfide bonds. The other domain is rich in β‐sheet (the β‐domain), has Cys 61‐Cys 77 and Cys 73‐Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine α‐lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three‐disulfide form, having a reduced Cys 6‐Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact α‐lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. The two‐disulfide form of α‐lactalbumin, having reduced Cys 6‐Cys 120 and Cys 28‐Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact α‐lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, α‐lactalbumin retains its calcium‐binding β‐domain, whereas the α‐domain is unfolded. It appears that the β‐domain does not require α‐domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded α‐domain.
π‐Turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence
Authors: Rajashankar K.R. , Ramakumar S.
Published: July 2010

The i + 5 → i hydrogen bonded turn conformation (7r‐turn) with the fifth residue adopting αL conformation is frequently found at the C‐terminus of helices in proteins and hence is speculated to be a “helix termination signal.” An analysis of the occurrence of i + 5 → i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C‐terminus), using coordinates of 228 protein crystal structures determined by X‐ray crystallography to better than 2.5 Å resolution is reported in this paper. Of 486 detected π‐turn conformations, 367 have the (i + 4)th residue in αL conformation, generally occurring at the C‐terminus of α‐helices, consistent with previous observations. However, a significant number (111) of π‐turn conformations occur with (i + 4)th residue in αR conformation also, generally occurring in α‐helices as distortions either at the terminii or at the middle, a novel finding. These two sets of 7r‐turn conformations are referred to by the names παL and παR‐turns, respectively, depending upon whether the (i + 4)th residue adopts αL or αR conformations. Four π‐turns, named π'αL ‐turns, were noticed to be mirror images of παL‐turns, and four more π‐turns, which have the (i + 4)th residue in β conformation and denoted as πβ‐turns, occur as a part of hairpin bend connecting twisted β‐strands. Consecutive π‐turns occur, but only with παR‐turns. The preference for amino acid residues is different in παL and παR‐turns. However, both show a preference for Pro after the C‐termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of παL‐turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in παL‐turns is mainly Gly and less often Asn. Although παR‐turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for παR‐turns, whereas good helix former Ala is not preferred. π‐Turns in peptides provide a picture of the π‐turn at atomic resolution. Only nine peptide‐based π‐turns are reported so far, and all of them belong to παL‐turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.
Chimeric hemoglobins — hybrids of human and swine hemoglobin: Assembly and stability of interspecies hybrids
Authors: Janardhan Rao M. , Seetharama Acharya A. , Manjula Belur N. , Kumar Ramesh
Published: July 2010

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely Hα2P
β
2 (H = human, P = swine). In an attempt to establish whether the absence of the swine α and human β (P
α
2H
β
2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine α and human β chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine α human β chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine α human β and the human α swine β interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P
α chain of P
α
2H
β
2 hybrid occurs in the presence of H
α chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P
α
2H
β
2 hybrid is lower compared to that of HbA. In contrast, P
β chain of H
α
2P
β
2 hybrid is refractory to exchange with H
β chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H
α
2P
β
2 is higher compared to that of HbA (H
α
2H
β
2). The swine α human β chimeric Hb undergoes subunit exchange reaction with human α‐chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra‐ and interdimeric interactions of the swine α and human β chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P
αH
β heterodimer (compared to the homodimers H
α
H
β and P
α
P
β)facilitates its segregation into the homodimers by subunit exchange reaction involving either H
α or P
β. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P
α
2
H
β
2 hybrid in transgenic swine expressing HbA.
An unusual route to thermostability disclosed by the comparison ofthermus thermophilusandescherichia coliinorganic pyrophosphatases
Authors: Salminen Tuna , Teplyakov Alexei , Kankare Jussi , Cooperman Barry S. , Lahti Reijo , Goldman Adrian
Published: July 2010

The structures ofEscherichia colisoluble inorganic pyrophosphatase (E‐PPase) andThermus thermophilussoluble inorganic pyrophosphatase (T‐PPase) have been compared to find the basis for the superior thermostability of T‐PPase. Both enzymes are D3hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T‐PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C‐terminal part of the protein, and more long‐range ion pairs from the C‐terminal helix to the rest of the molecule. Apart from the first five residues, the three‐dimensional structures of E‐PPase and T‐PPase monomers are very similar.

The one striking difference, however, is in the oligomeric interactions. In comparison with an E‐PPase monomer, each T‐PPase monomer is skewed by about 1 Å in thexyplane, is 0.3 Å closer to the center of the hexamer in thezdirection, and is rotated by approximately 7° about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid‐body motion has the effect that the hexamer is more tightly packed in T‐PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T‐PPase over E‐PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid‐body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.
Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases
Authors: Kuszewski John , Gronenborn Angela M. , Clore G. Marius
Published: July 2010

A new conformational database potential involving dihedral angle relationships in databases of high‐resolution highly refined protein crystal structures is presented as a method for improving the quality of structures generated from NMR data. The rationale for this procedure is based on the observation that uncertainties in the description of the nonbonded contacts present a key limiting factor in the attainable accuracy of protein NMR structures and that the nonbonded interaction terms presently used have poor discriminatory power between highland low‐probability local conformations. The idea behind the conformational database potential is to restrict sampling during simulated annealing refinement to conformations that are likely to be energetically possible by effectively limiting the choices of dihedral angles to those that are known to be physically realizable. In this manner, the variability in the structures produced by this method is primarily a function of the experimental restraints, rather than an artifact of a poor nonbonded interaction model. We tested this approach with the experimental NMR data (comprising an average of about 30 restraints per residue and consisting of interproton distances, torsion angles, 3JHNα coupling constants, and 13C chemical shifts) used previously to calculate the solution structure of reduced human thioredoxin (Qin J, Clore GM, Gronenborn AM, 1994, Structure 2:503–522). Incorporation of the conformational database potential into the target function used for refinement (which also includes terms for the experimental restraints, covalent geometry, and nonbonded interactions in the form of either a repulsive, repulsive‐attractive, or 6–12 Lennard‐Jones potential) results in a significant improvement in various quantitative measures of quality (Ramachandran plot, side‐chain torsion angles, overall packing). This is achieved without compromising the agreement with the experimental restraints and the deviations from idealized covalent geometry that remain within experimental error, and the agreement between calculated and observed 1H chemical shifts that provides an independent NMR parameter of accuracy. The method is equally applicable to crystallographic refinement, and should be particular useful during the early stages of either an NMR or crystallographic structure determination and in cases where relatively few experimental restraints can be derived from the measured data (due, for example, to broad lines in the NMR spectra or to poorly diffracting crystals).
The proteins encoded by the rbs operon of escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA
Authors: Barroga Charlene F. , Zhang Huide , Wajih Nadeem , Bouyer James H. , Hermodson Mark A.
Published: July 2010

The nucleotide‐binding component of the high‐affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7–7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5′‐FSBA and 8‐azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low‐level ATPase activity with a Km of about 140 μM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.
Substrate‐induced conformational changes in yeast 3‐phosphoglycerate kinase monitored by fluorescence of single tryptophan probes
Authors: Cheung Chia‐Wei , Mas Maria T.
Published: July 2010

3‐Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3‐phosphoglycerate (3‐PG) and ATP to 1,3‐diphosphoglycerate (1,3‐diPG) and ADP in the presence of magnesium ions. PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft. The large distance between the binding sites for 3‐PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge‐bending domain motion from open to closed conformation during catalysis. However, no direct experimental evidence exists for the “closed” conformation in the presence of both substrates. In this study, several PGK mutants with single tryptophans placed in various locations were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3‐PG, 1,3‐diPG, ADP, ATP, and PNP‐AMP (nonhydrolyzable analogue of ATP), and by 3‐PG and PNP‐AMP together. The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate‐induced conformational changes. Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains. A combination of both substrates generated much larger fluorescence changes than the individual substrates. The binding constants were determined for each substrate using probes situated in different locations.
Solution structure of a unique C5a semi‐synthetic antagonist: Implications in receptor binding
Authors: Zhang Xiaolu , Boyar William , Gonnella Nina C. , Galakatos Nicholas
Published: July 2010

The tertiary structure of a unique C5a receptor antagonist was determined by two‐dimensional NMR spectroscopy. The core domain of this 8‐kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)‐C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C‐terminal D‐Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D‐Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.
Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases
Authors: Marco Stefania Di , Fendrich Gabriele , Knecht Rene , Strauss Andre , Pohlig Gabriele , Heim Jutta , Priestle John P. , Grütter Markus G. , Sommerhoff Christian P.
Published: July 2010

A synthetic gene coding for the 55‐amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast α‐factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N‐terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N‐terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 Å and 2.4 Å, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.
Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala‐activated protein C mediated by factor Va
Authors: Gale Andrew J. , Sun Xi , Heeb Mary J. , Griffin John H.
Published: July 2010

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and Villa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the “activated” S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma‐derived and wild‐type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R α1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half‐life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506‐FVa compared with normal Arg 506‐FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.
Analysis of the structure and stability of omega loop A replacements in yeast iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Horner Scott R. , Oehrl Wolf , Schaak Diane L. , Boose Terry L. , Burton Randall E.
Published: July 2010

Omega (Ω)‐loop A, residues 18–32 in wild‐type yeast iso‐1‐cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low‐temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native‐like cytochrome c conformation, but are less stable than the wild‐type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single‐site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.