bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of the binding frame within a physiological ligand for the chaperone SecB
Authors: Topping Traci B. , Randall Linda L. 
Published: July 2010

AbstractThe 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

AbstractPrevious 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

AbstractWe 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

AbstractSerine 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

AbstractThe 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

AbstractWe 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 spectrometerTyrosine‐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 phosphopeptidesWe 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

AbstractDNA 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

AbstractWe 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

AbstractMonoTIM 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

AbstractThe 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

AbstractThe 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

AbstractA 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

AbstractThree‐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

AbstractAdenylosuccinate 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

AbstractAlthough 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

Abstractα‐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.