bluuebunny/crossref_metadata_2025_split huggingface dataset:
A cavity‐forming mutation in insulin induces segmental unfolding of a surrounding α‐helix
Authors: Xu Bin , Hua Qing‐Xin , Nakagawa Satoe H. , Jia Wenhua , Chu Ying‐Chi , Katsoyannis Panayotis G. , Weiss Michael A. 
Published: October 2010

AbstractTo investigate the cooperativity of insulin's structure, a cavity‐forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, IleA2→Ala in the A1–A8 α‐helix, does not impair disulfide pairing between chains. In accord with past studies of cavity‐forming mutations in globular proteins, a decrement was observed in thermodynamic stability (ΔΔGu 0.4–1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated α‐helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1–A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity‐forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of IleA2, a residue of low helical propensity, stabilizes the A1–A8 α‐helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The AlaA2 analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1–A8 α‐helix in receptor recognition.
Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies
Authors: Yuan Tao , Vogel Hans J. 
Published: October 2010

AbstractCalmodulin (CaM) is a 148‐residue regulatory calcium‐binding protein that activates a wide range of target proteins and enzymes. Calcium‐saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two “active sites” of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium‐dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM‐binding domain of myosin light‐chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild‐type CaM (WT‐CaM). One‐and two‐dimensional NMR studies of the Eth‐CaM and Nle‐CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth‐CaM and Nle‐CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the KD for Nle‐CaM also increased 3.5‐fold compared with WT‐CaM. These data show that the “active site” Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site‐directed mutagenesis studies of the Met residues of CaM.
KM+, a mannose‐binding lectin from artocarpus integrifolia: Amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the β‐prism fold
Authors: Rosa José César , Greene Lewis Joel , De Oliveira Paulo SÉRgio Lopes , Garratt Richard , Beltramini Leila , Resing Katheryn , Roque‐Barreira Maria‐Cristina 
Published: October 2010

AbstractThe complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl‐blocked N‐terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine‐rich linker that unites the regions homologous to the α‐and β‐chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (α‐D‐mannose, α‐D‐glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the β‐prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the β‐prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a β‐hairpin containing two classic‐type β‐bulges. We suggest the term β‐prism motif to describe this feature.
Relationships within the aldehyde dehydrogenase extended family
Authors: Perozich John , Nicholas Hugh , Wang Bi‐Cheng , Lindahl Ronald , Hempel John 
Published: October 2010

AbstractOne hundred‐forty‐five full‐length aldehyde dehydrogenase‐related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E‐NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the “Class 3” trunk, contains mostly substrate‐specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the “Class 102” trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate‐specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.
Thermodynamic linkage between the binding of protons and inhibitors to HIV‐1 protease
Authors: Trylska Joanna , Antosiewicz Jan , Geller Maciej , Hodge C. Nicholas , Klabe Ronald M. , Head Martha S. , Gilson Michael K. 
Published: October 2010

AbstractThe aspartyl dyad of free HIV‐1 protease has apparent pKas of ∼3 and ∼6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI‐272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson‐Boltzmann model of electrostatics is used to compute the apparent pKas of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns ϵ = 4 to the solute interior and uses a detailed model of ionization; the other uses ϵ = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pKas of ∼3 and ∼6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pKas in complex protein environments.Because binding of a cyclic urea inhibitor shifts the pKas of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.
Folding simulation of protein models on the structure‐based cubo‐octahedral lattice with the Contact Interactions algorithm
Authors: Toma Lucio , Toma Salvatore 
Published: October 2010

AbstractComputer simulations of protein models on lattices have been widely used as an aid in the study of protein folding process. Following the suggestion of Raghunathan and Jernigan (1997, Protein Sci 6:2072—2083) that the cubooctahedral lattice can allow a more realistic representation of proteins than other lattices, we propose here the use of a new set of internal coordinates θ for the description of a protein model on this lattice. An easy procedure for the conversion of the θ coordinates to the Cartesian coordinates is also described. When the Contact Interaction approach, already proposed by us for simulations on square or cubic lattices, was applied to the cubo‐octahedral lattice, the system obeyed the correct thermodynamics derived from the definition of energy. Thus, lattice simulations of protein models, in which secondary structure elements such as α‐helices or β‐strands can be easily identifiable, can be performed.
A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions
Authors: Kaushik Jai K. , Bhat Rajiv 
Published: October 2010

AbstractThe stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in‐depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico‐chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 °C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, ΔCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico‐chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.
A near‐native state on the slow refolding pathway of hen lysozyme
Authors: Kulkarni Sunita K. , Ashcroft Alison E. , Radford Sheena E. , Carey Michael , Masselos Dimitris , Robinson Carol V. 
Published: October 2010

AbstractThe refolding of four disulfide lysozyme (at pH 5.2, 20 °C) involves parallel pathways, which have been proposed to merge at a near‐native state. This species contains stable structure in the α‐and β‐domains but lacks a functional active site. Although previous experiments have demonstrated that the near‐native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 °C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths β‐domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near‐native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.
The signal transducer gp130: Solution structure of the carboxy‐terminal domain of the cytokine receptor homology region
Authors: Kernebeck Thomas , Pflanz Stefan , Heinrich Peter C. , Wollmer Axel , Grötzinger Joachim , Müller‐Newen Gerhard , Kurapkat Günther , Grzesiek Stephan , Scheek Ruud M. , Dijkstra Klaas 
Published: October 2010

AbstractThe transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin‐6‐type cytokines. It is a member of the cytokine‐receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine‐binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three‐dimensional structure of the carboxy‐terminal domain of this region was determined by multidimensional NMR. The domain consists of seven β‐strands constituting a fibronectin type III‐like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.
Protein‐protein recognition: An experimental and computational study of the R89K mutation in Raf and its effect on Ras binding
Authors: Zeng Jun , Simonson Thomas , Fridman Masha , Maruta Hiroshi , Treutlein Herbert R. 
Published: October 2010

AbstractBinding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras–Raf binding affinity. We have investigated experimentally and computationally the Raf‐R89K mutation, which abolishes signaling in vivo. The binding to [γ‐35S]GTP‐Ras of a fusion protein between the Raf‐binding domain (RBD) of Raf and GST was reduced at least 175‐fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9 &pm; 1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein–protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 Å away, namely residues 37–41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2–3 specific water molecules. This analysis will provide insights into the large experimental database of Ras–Raf mutations.
Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes
Authors: Bunik Victoria , Raddatz Günter , Bisswanger Hans , Lemaire Stephane , Meyer Yves , Jacquot Jean‐Pierre 
Published: October 2010

AbstractThe thioredoxin action upon the 2‐oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild‐type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin‐dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site‐directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2‐oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the α3/310 and a1 helices, the length of the α1 helix and the charges in the α2‐β3 and β4‐β5 linkers are found to correlate with the protein influence on the 2‐oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13–28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2‐oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.
Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non‐native secondary structure propensities
Authors: Cregut David , Serrano Luis 
Published: October 2010

AbstractThe usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close–to–wild‐type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central α‐helix by the sequence of the second β‐hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one‐third of the secondary structure has been replaced by a non‐native one. Models of the mutants were built based on the three‐dimensional structure of the wild‐type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild‐type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.
Sequence specificity, statistical potentials, and three‐dimensional structure prediction with self‐correcting distance geometry calculations of β‐sheet formation in proteins
Authors: Zhu Hongyao , Braun Werner 
Published: October 2010

AbstractA statistical analysis of a representative data set of 169 known protein structures was used to analyze the specificity of residue interactions between spatial neighboring strands in β‐sheets. Pairwise potentials were derived from the frequency of residue pairs in nearest contact, second nearest and third nearest contacts across neighboring β‐strands compared to the expected frequency of residue pairs in a random model. A pseudo‐energy function based on these statistical pairwise potentials recognized native β‐sheets among possible alternative pairings. The native pairing was found within the three lowest energies in 73% of the cases in the training data set and in 63% of β‐sheets in a test data set of 67 proteins, which were not part of the training set. The energy function was also used to detect tripeptides, which occur frequently in β‐sheets of native proteins. The majority of native partners of tripeptides were distributed in a low energy range. Self‐correcting distance geometry (SECODG) calculations using distance constraints sets derived from possible low energy pairing of β‐strands uniquely identified the native pairing of the β‐sheet in pancreatic trypsin inhibitor (BPTI). These results will be useful for predicting the structure of proteins from their amino acid sequence as well as for the design of proteins containing β‐sheets.
Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopy
Authors: Sreerama Narasimha , Venyaminov Sergei Y.U. , Woody Robert W. 
Published: October 2010

AbstractA simple approach to estimate the number of α‐helical and β‐strand segments from protein circular dichroism spectra is described. The α‐helix and β‐sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given α‐helix or β‐strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per α‐helix and two residues per β‐strand may be considered distorted in proteins. The number of α‐helical and β‐strand segments and their average length in a given protein were estimated from the fraction of distorted α‐helix and β‐strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.