bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of glycine (residue 89) in the central helix of EF‐hand protein troponin‐C exposed following amino‐terminal α‐helix deletion
Authors: Ding Xiao‐Ling , Akella Árvind Babu , Su Hong , Gulati Jagdish 
Published: February 2009

AbstractBecause an N‐terminal α‐helical (N‐helix) arm and a KGK‐triplet (residues 88KGK90) in the central helix of troponin‐C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure‐function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N‐helix and the KGK‐triplet in TnC. Noticeably, the amino acid compositions vary between the N‐helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK‐triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N‐helix (mutant ΔNt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90‐triplet (mutant ΔNtΔKGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685–11690). Using this functional test, we find that replacement of Gly‐89 with a Leu or an Ala could also overcome the contractile defect associated with N‐helix deletion. On the other hand, replacement of the skeletal TnC N‐helix with cardiac type N‐helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly‐89 residue in skeletal TnC and suggest that the N‐terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N‐helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.
Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease
Authors: Xie Dong , Freire Ernesto , Fox Robert 
Published: February 2009

AbstractHigh‐sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117 → Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2‐state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the β subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0–100 °C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure‐based thermodynamic calculations also predict that the most probable intermediate is one in which the β subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.
Cavities and packing at protein interfaces
Authors: Hubbard Simon J. , Argos Patrick 
Published: February 2009

AbstractAn analysis of internal packing defects or “cavities” (both empty and water‐containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water‐filled “cavities” possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavity‐surrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and inter‐domain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water‐filled. We observe increased cavity volume at domain‐domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.
Hydrogen exchange in BPTI variants that do not share a common disulfide bond
Authors: Schulman Brenda A. , Kim Peter S. 
Published: February 2009

AbstractBovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30‐51, 5‐55, and 14‐38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5‐55]A]a and [30‐51; 14‐38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide‐bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native‐like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI foldiing intermediates containing either the 5‐55 or the 30‐51 disulfide bond.
The role of transmembrane domain III in the lactose permease of escherichia coli
Authors: Sahin‐Tóth Miklós , Frillingos Stathis , Bibi Eitan , Gonzalez Alberto , Ronald Kaback H. 
Published: February 2009

AbstractDeletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys‐scanning mutagenesis. Using a functional mutant devoid of Cys residues (C‐less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty‐one of the 25 mutants accumulate lactose to >70% of the steady‐state exhibited by C‐less permease, and an additional 3 mutants transport to lower, but significant levels (40–60% of C‐less). Cys replacement for Leu 76 results in low transport activity (18% of C‐less). However, when placed in the wild‐type background, mutant Leu 76 → Cys exhibits highly significant rates of transport (55% of wild type) and steady‐state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N‐ethylmaleimide show that mutant Gly 96 → Cys is rapidly inactivated, whereas the other single‐Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96 → Cys permease is enhanced at least 2‐fold in the presence of β‐galactopyranosyl 1‐thio‐β,D‐galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.
Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin
Authors: Wills Fiona L. , Mccubbin William D. , Kay Cyril M. , Gimona Mario , Strasser Peter 
Published: February 2009

AbstractNative calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino‐terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2–51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl‐terminal truncated mutant of calponin comprising residues 1–228 (CP 1–228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1–228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1–228 in a Ca2+‐dependent fashion, indicating that there is a second site of interaction between residues 52–228. Temperature denaturation of the carboxyl‐terminal truncated fragment compared with whole calponin show that the carboxyl‐terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45–228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.
Dissecting the energetics of an antibody‐antigen interface by alanine shaving and molecular grafting
Authors: Jin Lei , Wells James A. 
Published: February 2009

AbstractAlanine‐scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851–865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7–16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10‐fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6‐ to >500‐fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10‐fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4‐helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony‐stimulating factor, were unsuccessful. These studies show that only a small number of side chains on hGH confers tight binding affinity to MAb3 and that substituting or transplanting them onto another scaffold has narrow design tolerances.
Determinants of protein side‐chain packing
Authors: Tanimura Ryuji , Kidera Akinori , Nakamura Haruki 
Published: February 2009

AbstractThe problem of protein side‐chain packing for a given backbone trace is investigated using 3 different prediction models. The first requires an exhaustive search of all possible combinations of side‐chain conformers, using the dead‐end elimination theorem. The second considers only side‐chain‐backbone interactions, whereas the third neglects side‐chain‐backbone interactions and instead keeps side‐chain‐side‐chain interactions. Predictions of side‐chain conformations for 11 proteins using all 3 models show that removal of side‐chain‐side‐chain interactions does not cause a large decrease in the prediction accuracy, whereas the model having only side‐chain‐side‐chain interactions still retains a significant level of accuracy. These results suggest that the 2 classes of interactions, side‐chain‐backbone and side‐chain‐side‐chain, are consistent with each other and work concurrently to stabilize the native conformations. This is confirmed by analyses of energy spectra of the side‐chain conformations derived from the fourth prediction model, the Independent model, which gives almost the same quality of the prediction as the dead‐end elimination. The analyses indicate that the 2 classes of interactions simultaneously increase the energy difference between the native and nonnative conformations.
Molecular evolution and domain structure of plasminogen‐related growth factors (HGF/SF and HGF1/MSP)
Authors: Donate Luis Enrique , Srinivasan N. , Sowdhamini R. , Gherardi Ermanno , Blundell Tom L. , Aparicio Samuel 
Published: February 2009

AbstractPlasminogen‐related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor‐like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron–exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N‐terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide‐bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix‐strand‐helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three‐dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine‐binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.
Conservation of polyproline II helices in homologous proteins: Implications for structure prediction by model building
Authors: Adzhubei Alexei A. , Sternberg Michael J.E. 
Published: February 2009

AbstractLeft‐handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4–8 residues. This paper analyzes the structural conservation of PPII‐helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII‐helices, the α‐helices, and the β‐strands. The PPII‐helices are consistently conserved at the level of 100–80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 Å. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII‐helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII‐helices, α‐helices, and β‐strands. The PPII‐helices are shown to participate in supersecondary elements such as PPII‐helix/α‐helix. The conservation of PPII‐helices depends on the conservation of a supersecondary element as a whole. PPII‐helices also form links, possibly flexible, in the interdomain regions. The role of the PPII‐helices in model building by homology is 2‐fold: they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.
High‐Sensitivity sequencing of large proteins: Partial structure of the rapamycin‐fkbp12 target
Authors: Erdjument‐Bromage Hediye , Lui Mary , Tempst Paul , Sabatini David M. , Snyder Solomon H. 
Published: February 2009

AbstractWe report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher‐sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose‐bound proteins; and (3) accurate UV spectroscopic identification of Trp‐ and Cys‐containing peptides. In addition, we describe strategies for 2‐dimensional liquid chromatographic peptide isolation from complex mixtures and a multi‐analytical approach to peptide sequence analysis (Edman sequencing, matrix‐assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies were applied in tandem to the primary structural analysis of a gel‐purified, 250‐kDa protein (mammalian target of rapamycin‐FKBP12 complex), available in low picomolar quantities only. More than 300‐amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well‐characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.
Crystallization and preliminary x‐ray analysis of glucose‐fructose oxidoreductase from zymomonas mobilis
Authors: Loos Heidi , Ermler Ulrich , Sprenger Georg A. , Sahm Hermann 
Published: February 2009

AbstractGlucose‐fructose oxidoreductase (E.C. 1.1.99.‐) from the ethanol‐producing Gram‐negative bacterium Zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotet‐ramer of 160 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P21212, with unit cell constants of 84.6 Å, 94.1 Å, and 117.0 Å, consistent with two monomers in the asymmetric unit. They diffract to a resolution of about 2 Å and are suitable for X‐ray structure determination.
The astacin family of metalloendopeptidases
Authors: Bond Judith S. , Beynon Robert J. 
Published: February 2009

AbstractThe astacin family of metalloendopeptidases was recognized as a novel family of proteases in the 1990s. The crayfish enzyme astacin was the first characterized and is one of the smallest members of the family. More than 20 members of the family have now been identified. They have been detected in species ranging from hydra to humans, in mature and in developmental systems. Proposed functions of these proteases include activation of growth factors, degradation of polypeptides, and processing of extracellular proteins. Astacin family proteases are synthesized with NH2‐terminal signal and proenzyme sequences, and many (such as meprins, BMP‐1, tolloid) contain multiple domains COOH‐terminal to the protease domain. They are either secreted from cells or are plasma membrane‐associated enzymes. They have some distinguishing features in addition to the signature sequence in the protease domain: HEXXHXXGFXHEXXRXDR. They have a unique type of zinc binding, with pentacoordination, and a protease domain tertiary structure that contains common attributes with serralysins, matrix metalloendopeptidases, and snake venom proteases; they cleave peptide bonds in polypeptides such as insulin B chain and bradykinin and in proteins such as casein and gelatin; and they have arylamidase activity. Meprins are unique proteases in the astacin family, and indeed in the animal kingdom, in their oligomeric structure; they are dimers of disulfide‐linked dimers and are highly glycosylated, type I integral membrane proteins that have many attributes of receptors or integrins with adhesion, epidermal growth factor‐like, and transmembrane domains. The α and β subunits are differentially expressed and processed to yield latent and active proteases as well as membrane‐associated and secreted forms. Meprins represent excellent models of hetero‐ and homo‐oligomeric enzymes that are regulated at the transcriptional and posttranslational levels.
High‐resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer
Authors: Abele U. , Schulz G.E. 
Published: February 2009

AbstractThe structure of adenylate kinase from yeast ligated with the two‐substrate‐mimicking inhibitor Ap5A and Mg2+ has been refined to 1.96 Å resolution. In addition, the refined structure of the same complex with a bound imidazole molecule replacing Mg2+ has been determined at 1.63 Å. These structures indicate that replacing Mg2+ by imidazole disturbs the water structure and thus the complex. A comparison with the G‐proteins shows that Mg2+ is exactly at the same position with respect to the phosphates. However, although the Mg2+ ligand sphere of the G‐proteins is a regular octahedron containing peptide ligands, the reported adenylate kinase has no such ligands and an open octahedron leaving space for the Mg2+ to accompany the transferred phosphoryl group. A superposition of the known crystalline and therefore perturbed phosphoryl transfer geometries in the adenylate kinases demonstrates that all of them are close to the start of the forward reaction with bound ATP and AMP. Averaging all observed perturbed structures gives rise to a close approximation of the transition state, indicating in general how to establish an elusive transition state geometry. The average shows that the in‐line phosphoryl transfer is associative, because there is no space for a dissociative metaphosphate intermediate. As a side result, the secondary dipole interaction in the α‐helices of both protein structures has been quantified.