bluuebunny/crossref_metadata_2025_split huggingface dataset:
A role for surface hydrophobicity in protein‐protein recognition
Authors: Young L. , Jernigan R.L. , Covell D.G. 
Published: July 2010

AbstractThe role of hydrophobicity as a determinant of protein‐protein interactions is examined. Surfaces of apo‐protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol‐binding protein, and their associated ligands with available X‐ray structures for their complexed forms, are scanned to determine clusters of surface‐accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co‐crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one‐third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well‐defined loci for possible ligand attachment.
A β‐turn in α‐amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies
Authors: Baumann Karlheinz , Zanotti Giancarlo , Faulstich Heinz 
Published: July 2010

AbstractFour amatoxin‐binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 α‐amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X‐ray analysis to be part of a β‐turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17: 3790‐3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the β‐turn is likewise important. In addition to the β‐turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6′‐hydroxy‐Trp4 and OH of hydroxy‐Pro2 and dihydroxy‐Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6″hydroxy‐Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X‐ray data by providing information on the amount of binding energy contributed by single structural elements.
Structure of glutathione reductase from escherichia coli at 1.86 Å resolution: Comparison with the enzyme from human erythrocytes
Authors: Mittl Peer R.E. , Schulz Georg E. 
Published: July 2010

AbstractThe crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R‐factor of 16.8% at 1.86 Å resolution. The molecular 2‐fold axis of the dimer is local but very close to a possible crystallographic 2‐fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well‐known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel β‐sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu‐Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.
Biological meaning, statistical significance, and classification of local spatial similarities in nonhomologous proteins
Authors: Alexandrov Nickolai N. , Gō Nobuhiro 
Published: July 2010

AbstractWe have completed an exhaustive search for the common spatial arrangements of backbone fragments (SARFs) in nonhomologous proteins. This type of local structural similarity, incorporating short fragments of backbone atoms, arranged not necessarily in the same order along the polypeptide chain, appears to be important for protein function and stability. To estimate the statistical significance of the similarities, we have introduced a similarity score. We present several locally similar structures, with a large similarity score, which have not yet been reported. On the basis of the results of pairwise comparison, we have performed hierarchical cluster analysis of protein structures. Our analysis is not limited by comparison of single chains but also includes complex molecules consisting of several subunits. The SARFs with backbone fragments from different polypeptide chains provide a stable interaction between subunits in protein molecules. In many cases the active site of enzymes is located at the same position relative to the common SARFs, implying a function of the certain SARFs as a universal interface of the protein‐substrate interaction.
Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme
Authors: Goldberg Michel E. , Guillou Yvonne 
Published: July 2010

AbstractTo assess the respective roles of local and long‐range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous‐flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far‐UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the final conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.
Folding of the multidomain human immunodeficiency virus type‐I integrase
Authors: Grandgenett Duane P. , Goodarzi Goodarz 
Published: July 2010

AbstractProtein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine · HCl–5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine · HCl allowed for wide variation of protein concentration in the folding reaction. A 6‐fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3′ OH end processing, strand transfer, and disintegration using various human immunodeficiency virus‐1 (HIV‐1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers were produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is indeterminate. Our results suggest that monomers of the multidomain HIV‐1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.
Monte Carlo study of the effect of β2‐microglobulin on the binding cleft of the HLA‐A2 complex
Authors: Bouzida Djamal , Garnier Jean , Brower Richard , Cornette James , Delisi Charles 
Published: July 2010

AbstractPeptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β2‐microglobulin. We present results of Monte Carlo simulations of the β‐pleated sheet floor of the human class I MHC molecule, HLA‐A2, with and without β2‐microglobulin. We find a significant effect of β2‐microglobulin on the side chains of residues near a region that would accommodate the C‐terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β2‐microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.
Harmonic and anharmonic aspects in the dynamics of BPTI: A normal mode analysis and principal component analysis
Authors: Hayward Steven , Kitao Akio , Gō Nobuhiro 
Published: July 2010

AbstractA comparison is made between a 200‐ps molecular dynamics simulation in vacuum and a normal mode analysis on the protein bovine pancreatic trypsin inhibitor (BPTI) in order to elucidate the dual aspects of harmonicity and anharmonicity in the dynamics of proteins. The molecular dynamics trajectory is analyzed using principal component analysis, an effective harmonic analysis suited for comparison with the results from the normal mode analysis. The results suggest that the first principal component shows qualitatively different behavior from higher principal components and is associated with apparent barrier crossing events on an anharmonic conformational energy surface. The higher principal components appear to have probability distributions that are well approximated by Gaussians, indicating harmonicity. Eliminating the contribution from the first principal component reveals a great deal of correspondence between the 2 methods. This correspondence, however, involves a factor of 2, as the variances of the distributions of the higher principal components are, on average, roughly twice those found from the normal mode analysis. A model is proposed to reconcile these results with those from previous analyses.
Thermodynamics of staphylococcal nuclease denaturation. I. The acid‐denatured state
Authors: Carra John H. , Anderson Elizabeth A. , Privalov Peter L. 
Published: July 2010

AbstractUsing high‐sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower ΔCp and ΔH for the denaturation reaction. The acid‐denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid‐denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of ΔH on pH.
A 70‐amino acid zinc‐binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer
Authors: Zhou Bin‐Bing , Waldrop Grover L. , Lum Lawrence , Schachman H. K. 
Published: July 2010

AbstractInteraction between a 70‐amino acid and zinc‐binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady‐state kinetics, heavy‐atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N‐(phosphonacetyl)‐L‐aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain‐C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased “stickiness” of carbamoyl phosphate to the Zn domain‐C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain‐C trimer complex was about 10–2 that from C trimer. Additional evidence for a change in binding of carbamoyl phosphate as a result of interaction of zinc domain and C trimer was obtained from the pH profile for Kia for carbamoyl phosphate. Whereas participation of only 1 protonated residue is implicated in binding of carbamoyl phosphate to C trimer, 2 residues must be ionized for binding to the Zn domain‐C trimer complex.
Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase
Authors: Rochet Jean‐Christophe , Bridger William A. 
Published: July 2010

AbstractThe enzyme CoA transferase (succinyl‐CoA:3‐ketoacid coenzyme A transferase [3‐oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active‐site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme‐CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS‐PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C‐terminal fragment is blocked at its N‐terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N‐terminus corresponding to the site of proteolysis and a C‐terminus determined by the site of autolysis. This peptide was purified by reversed‐phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active‐site structure and catalytic mechanism of CoA transferase.
Interleukin‐1βconverting enzyme: A novel cysteine protease required for IL‐1β production and implicated in programmed cell death
Authors: Thornberry Nancy A. , Molineaux Susan M. 
Published: July 2010

AbstractInterleukin‐1β converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL‐1β, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED‐3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.
Differentiation of peptide molecular recognition by phospholipase Cγ‐1 Src homology‐2 domain and a mutant Tyr phosphatase PTP1bC215S
Authors: Maclean Derek , Sefler Andrea M. , Zhu Guochang , Decker Stuart J. , Saltiel Alan R. , Singh Juswinder , Mcnamara Dennis , Dobrusin Ellen M. , Sawyer Tomi K. 
Published: July 2010

AbstractActivated epidermal growth factor receptor (EGFR) undergoes autophosphorylation on several cytoplasmic tyrosine residues, which may then associate with the src homology‐2 (SH2) domains of effector proteins such as phospholipase Cγ‐1 (PLCγ‐1). Specific phosphotyrosine (pTyr)‐modified EGFR fragment peptides can inhibit this intermolecular binding between activated EGFR and a tandem amino‐ and carboxy‐terminal (N/C) SH2 protein construct derived from PLCγ‐1. In this study, we further explored the molecular recognition of phosphorylated EGFR988–998 (Asp‐Ala‐Asp‐Glu‐pTyr‐Leu‐Ile‐Pro‐Gln‐Gln‐Gly, I) by PLCγ‐1 N/C SH2 in terms of singular Ala substitutions for amino acid residues N‐ and C‐terminal to the pTyr (P site) of phosphopeptide I. Comparison of the extent to which these phosphopeptides inhibited binding of PLCγ‐1 N/C SH2 to activated EGFR showed the critical importance of amino acid side chains at positions P+2 (Ile994), P+3 (Pro995), and P+4 (Gln996). Relative to phosphopeptide 1, multiple Ala substitution throughout the N‐terminal sequence, N‐terminal truncation, or dephosphorylation of pTyr each resulted in significantly decreased binding to PLCγ‐1 N/C SH2. These structure‐activity results were analyzed by molecular modeling studies of the predicted binding of phosphopeptide 1 to each the N‐ and C‐terminal SH2 domains of PLCγ‐1. In the case of the PLCγ‐1 C‐terminal SH2, the coordinates of a phosphopeptide SH2 domain complex recently determined by NMR studies (Pascal SM, et al., 1994, Cell 77:461–472) were used directly, whereas the N‐terminal SH2 domain model was developed from homology model building using the NMR coordinates of the PLCγ‐1 C‐terminal SH2 as a template. Relative to PLCγ‐1 N/C SH2 domain molecular recognition, the binding affinities of phosphorylated EGFR988–998 and its Ala‐scan analog series to a catalytically inert mutant protein tyrosine phosphatase, PTPlbC215S, showed only a slight loss in binding at the P+1 (Leu) site. Therefore, peptide molecular recognition by two functionally distinct signal transduction proteins, PLCγ‐1 and PTP1b, may be differentiated in terms of systematic chemical modifications of EGFR988_998 and comparative structure‐activity analysis.
Thermodynamics of heme‐induced conformational changes in hemopexin: Role of domain‐domain interactions
Authors: Wu Ming‐Lei , Morgan William T. 
Published: July 2010

AbstractHemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor‐mediated endocytosis. A hinge region connects the two non‐disulfide‐linked domains of hemopexin, a 35‐kDa N‐terminal domain (domain I) that binds heme, and a 25‐kDa C‐terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo‐hemopexin and another in heme‐hemopexin, and this change is important in facilitating the association of heme‐hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein‐protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 μM and 55 μM, respectively. The interaction between heme‐domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (ΔCP) of ‐720 cal/mol‐K, and a calculated entropy of +65 cal/mol‐K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (ΔH) of ‐3.6 kcal/mol and change in entropy (AS) of +8.1 cal/mol‐K for the association of apo‐domain I with domain II was obtained. The association is, however, not strongly dependent on ionic strength. The values of the thermodynamic parameters obtained suggest that different types of interactions predominate in the presence and absence of heme: hydrophobic interactions between heme‐domain I and domain II and likely a balanced mix of different forces (e.g., van der Waals and hydrophobic interactions) between apodomain I and domain II.
Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation
Authors: Hurley John K. , Caffrey Michael S. , Markley John L. , Cheng Hong , Xia Bin , Chae Young Kee , Holden Hazel M. , Tollin Gordon 
Published: July 2010

AbstractThe stability properties of oxidized wild‐type (wt) and site‐directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn‐HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287–3295); we also find this to be true with respect to Gdn‐HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X‐ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities. Those F65 mutants showing diminished stability (i.e., F65A and F65I) have previously been shown (Hurley JK, et al., 1993b, Biochemistry 52:9346–9354) to be severely impaired kinetically in their electron transfer (ET) reaction with ferredoxin:NADP + reductase (FNR), a physiological reaction partner of Vfd. Mutants F65W and F65Y, which, as noted, are like wt in stability, also functioned like wt in the ET reaction with FNR (Hurley JK, et al., 1993a, J Am Chem Soc 115:11698–11701). Possible reasons for this correlation between ET properties of the F65 mutants and their conformational stabilities are discussed.