bluuebunny/crossref_metadata_2025_split huggingface dataset:
Competing modes of self‐association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization
Authors: Laederach Alain , Cradic Kendall W. , Brazin Kristine N. , Zamoon Jamillah , Fulton D. Bruce , Huang Xin‐Yun , Andreotti Amy H. 
Published: October 2010

AbstractA nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer‐dimer equilibria. The Btk fragment studied contains two consecutive proline‐rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino‐terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy‐terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino‐terminal proline stretch; the other, in the carboxy‐terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino‐terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined Ka values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co‐localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.
Removal of surface charge‐charge interactions from ubiquitin leaves the protein folded and very stable
Authors: Loladze Vakhtang V. , Makhatadze George I. 
Published: October 2010

AbstractThe contribution of solvent‐exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site‐directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys‐amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (ΔG = 18 kJ/mol). These results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.
Mechanics and dynamics of B1 domain of protein G: Role of packing and surface hydrophobic residues
Authors: Ceruso Marc A. , Amadei Andrea , Nola Alfredo Di 
Published: October 2010

AbstractThe structural organization of the B1 domain of streptococcal protein G (PGA) has been probed using molecular dynamics simulations, with a particular emphasis on the role of the solvent exposed Ile6 residue. In addition to the native protein (WT‐PGA), three single‐mutants (I6G‐PGA, I6F‐PGA, and I6T‐PGA), one double‐mutant (I6T, T53G‐PGA), and three isolated peptide fragments (corresponding to the helix and the two β‐hairpins) were studied in the presence of explicit water molecules. Comparative analysis of the various systems showed that the level of perturbation was directly related to the hydrophobicity and the size of the side chain of residue 6, the internal rigidity of the proteins decreasing in the order I6T‐PGA > I6G‐PGA > WT‐PGA > I6F‐PGA. The results emphasized the importance of residue 6 in controlling both the integrity of the sheet's surface and the orientation of the helix in relation to the sheet by modulation of surface/core interactions. The effects of mutations were delocalized across the structure, and glycine residues, in particular, absorbed most of the introduced strain. A qualitative structural decomposition of the native fold into elementary building‐blocks was achieved using principal component analysis and mechanical response matrices. Within this framework, internal motions of the protein were described as coordinated articulations of these structural units, mutations affecting mostly the amplitude of the motions rather than the structure/location of the building‐blocks. Analysis of the isolated peptidic fragments suggested that packing did not play a determinant role in defining the elementary building‐blocks, but that chain topology was mostly responsible.
Association of partially‐folded intermediates of staphylococcal nuclease induces structure and stability
Authors: Uversky Vladimir N. , Fink Anthony L. , Khurana Ritu , Karnoup Anton S. , Segel Daniel J. , Doniach Sebastian 
Published: October 2010

AbstractStaphylococcal nuclease forms three different partially‐folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (≤0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less‐ordered partially‐folded intermediates (A1andA2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small‐angle X‐ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.
Assignment of side‐chain conformation using adiabatic energy mapping, free energy perturbation, and molecular dynamic simulations
Authors: Frimurer Thomas M. , Olsen Ole Hvilsted , Peters Günther H. , Sørensen Morten Dahl , Led Jens JØRgen 
Published: October 2010

AbstractNMR spectroscopic analysis of the C‐terminal Kunitz domain fragment (α3(VI)) from the human α3‐chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X‐ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X‐ray crystallographic structure and solution structure of α3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free‐energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77 6 0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the α3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13‐Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp21 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.
The crystal structure of α‐thrombin‐hirunorm IV complex reveals a novel specificity site recognition mode
Authors: Lombardi Angela , Simone Giuseppina De , Nastri Flavia , Galdiero Stefania , Pedone Carlo , Pavone Vincenzo , Morte Rossella Della , Staiano Norma , Bolognesi Martino 
Published: October 2010

AbstractThe X‐ray crystal structure of the human α‐thrombin‐hirunorm IV complex has been determined at 2.5 Å resolution, and refined to an R‐factor of 0.173. The structure reveals an inhibitor binding mode distinctive of a true hirudin mimetic, which justifies the high inhibitory potency and the selectivity of hirunorm IV. This novel inhibitor, composed of 26 amino acids, interacts through the N‐terminal end with the α‐thrombin active site in a nonsubstrate mode, and binds specifically to the fibrinogen recognition exosite through the C‐terminal end. The backbone of the N‐terminal tripeptide Chg1″‐Arg2″‐2Nal3″ (Chg, cyclohexyl‐glycine; 2Nal, β‐(2‐naphthyl)‐alanine) forms a parallel β‐strand to the thrombin main‐chain segment Ser214—Gly216. The Chg1″ side chain occupies the S2 site, Arg2″ penetrates into the S1 specificity site, while the 2Nal3″ side chain occupies the aryl binding site. The Arg2″ side chain enters the S1 specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2″ side chain establishes the typical ion pair with the carboxylate group of Asp189.
Crystal structure of the FMN‐binding domain of human cytochrome P450 reductase at 1.93 Å resolution
Authors: Zhao Qiang , Modi Sandeep , Smith Graeme , Paine Mark , Mcdonagh Paul D. , Wolf C. Roland , Tew David , Lian Lu‐Yun , Roberts Gordon C.K. , Driessen Huub P.C. 
Published: October 2010

AbstractThe crystal structure of the FMN‐binding domain of human NADPH‐cytochrome P450 reductase (P450R‐FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 å resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997,J Biomol NMR 10:63–75) and to the corresponding domain in the 2.6 å crystal structure of intact rat P450R (Wang M et al., 1997,Proc Nat Acad Sci USA 94:8411–8416). The crystal structure of P450R‐FMN reported here confirms the overall similarity of its α‐β‐α architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central β‐sheet. The marked similarity between P450R‐FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R‐FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P450 2D6 and to cytochromec, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochromec.
Pair potentials for protein folding: Choice of reference states and sensitivity of predicted native states to variations in the interaction schemes
Authors: Betancourt Marcos R. , Thirumalai D. 
Published: October 2010

AbstractWe examine the similarities and differences between two widely used knowledge‐based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa–Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676–688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner.
Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state
Authors: Rankin Saffron E. , Watts Anthony , Roder Heinrich , Pinheiro Teresa J.T. 
Published: October 2010

AbstractUnfolded apocytochrome c acquires an α‐helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt‐induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid‐inserted helical conformation proceeds via a collapsed intermediate state (IC). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme‐free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of IC through a more extended state associated with the membrane surface (IS). While IC appears to be as compact as salt‐induced compact states in solution with substantial α‐helix content, the final lipid‐inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an α‐helix content which resembles that of native cytochrome c.
Breaking the low barrier hydrogen bond in a serine protease
Authors: Kidd Richard D. , Sears Pamela , Huang Dee‐Hua , Witte Krista , Wong Chi‐Huey , Farber Gregory K. 
Published: October 2010

AbstractThe serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water‐miscible organic co‐solvent such as N, N‐dimethylformamide (DMF). However, in 50% DMF, the kcat for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the kcat for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397 + 1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 å resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 1808 around the Cβ‐Cγ bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co‐solvents.
Role of the lateral channel in catalase HPII of Escherichia coli
Authors: Sevinc M. Serdal , Maté Maria J. , Switala Jack , Fita Ignacio , Loewen Peter C. 
Published: October 2010

AbstractThe heme‐containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N‐ and C‐terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585–590 of the C‐terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active‐site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 Å in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild‐type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 Å resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.
Comparison of protein–protein interactions in serine protease‐inhibitor and antibody‐antigen complexes: Implications for the protein docking problem
Authors: Jackson Richard M. 
Published: October 2010

AbstractThe protein‐protein interaction energy of 12 nonhomologous serine protease‐inhibitor and 15 antibody‐antigen complexes is calculated using a molecular mechanics formalism and dissected in terms of the main‐chain vs. side‐chain contribution, nonrotameric side‐chain contributions, and amino acid residue type involvement in the interface interaction.There are major differences in the interactions of the two types of protein‐protein complex. Protease‐inhibitor complexes interact predominantly through a main‐chain‐main‐chain mechanism while antibody‐antigen complexes interact predominantly through a side‐chain‐side‐chain or a side‐chain‐main‐chain mechanism. However, there is no simple correlation between the main‐chain‐main‐chain interaction energy and the percentage of main‐chain surface area buried on binding. The interaction energy is equally effected by the presence of nonrotameric side‐chain conformations, which constitute ∼20% of the interaction energy. The ability to reproduce the interface interaction energy of the crystal structure if original side‐chain conformations are removed from the calculation is much greater in the protease‐inhibitor complexes than the antibody‐antigen complexes. The success of a rotameric model for protein‐protein docking appears dependent on the extent of the main‐chain‐main‐chain contribution to binding.Analysis of (1) residue type and (2) residue pair interactions at the interface show that antibody‐antigen interactions are very restricted with over 70% of the antibody energy attributable to just six residue types (Tyr > Asp > Asn > Ser > Glu > Trp) in agreement with previous studies on residue propensity. However, it is found here that 50% of the antigen energy is attributable to just four residue types (Arg = Lys > Asn > Asp). On average just 12 residue pair interactions (6%) contribute over 40% of the favorable interaction energy in the antibody‐antigen complexes, with charge‐charge and charge/polar‐tyrosine interactions being prominent. In contrast protease inhibitors use a diverse set of residue types and residue pair interactions.
Actin surface structure revealed by antibody imprints: Evaluation of phage‐display analysis of anti‐actin antibodies
Authors: Jesaitis Algirdas J. , Gizachew Dawit , Dratz Edward A. , Siemsen Daniel W. , Stone Kim Clawson , Burritt James B. 
Published: October 2010

AbstractPhage‐display peptide library analysis of an anti‐F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X‐ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage‐display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti‐actin immune serum. One hundred and forty‐four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F‐actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F‐actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken β‐actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X‐ray crystal structure suggested that within a 15 å radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root‐mean‐square deviation fits of 2.1–2.5 å. We conclude that phage‐display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.
Genome analysis: Assigning protein coding regions to three‐dimensional structures
Authors: Salamov Asaf A. , Suwa Makiko , Orengo Christine A. , Swindells Mark B. 
Published: October 2010

AbstractWe describe the results of a procedure for maximizing the number of sequences that can be reliably linked to a protein of known three‐dimensional structure. Unlike other methods, which try to increase sensitivity through the use of fold recognition software, we only use conventional sequence alignment tools, but apply them in a manner that significantly increases the number of relationships detected. We analyzed 11 genomes and found that, depending on the genome, between 23 and 32% of the ORFs had significant matches to proteins of known structure. In all cases, the aligned region consisted of either >100 residues or >50% of the smaller sequence. Slightly higher percentages could be attained if smaller motifs were also included. This is significantly higher than most previously reported methods, even those that have a fold‐recognition component. We survey the biochemical and structural characteristics of the most frequently occurring proteins, and discuss the extent to which alignment methods can realistically assign function to gene products.