bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural determinants of trypsin affinity and specificity for cationic inhibitors
Authors: Polticelli Fabio , Honig Barry , Ascenzi Paolo , Bolognesi Martino 
Published: February 2009

AbstractThe binding free energies of four inhibitors to bovine β‐trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson‐Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy‐surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine‐ and lysine‐based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp 189 in the inhibitor‐free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor‐free state, thus reducing the entropie cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.
Structure of the fibrinogen γ‐chain integrin binding and factor XIIIa cross‐linking sites obtained through carrier protein driven crystallization
Authors: Ware Scott , Anderson Wayne F. , Donahue John P. , Hawiger Jacek 
Published: February 2009

AbstractThe human fibrinogen γ‐chain C‐terminal segment functions as the platelet integrin binding site as well as the Factor XIIIa cross‐linking substrate and thus plays an important role in blood clot formation and stabilization. The three‐dimensional structure of this segment has been determined using carrier protein driven crystallization. The C‐terminal segment, γ‐(398–411), was attached to a linker sequence at the C‐terminus of glutathione S‐transferase and the structure of this fusion protein determined at 1.8 Å resolution. Functional studies of the chimeric protein demonstrate that the fibrinogen sequence in the presence of the carrier protein retains its specific functions as ligand for platelet integrin αIIbβ3 (gpIIb/IIIa) and as a cross‐linking substrate for Factor XIIIa. The structure obtained for the fibrinogen γ‐chain segment is not affected by crystal packing and can provide the missing links to the recently reported model of cross‐linked fibrin.
Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus
Authors: Sakowicz Roman , Farlow Sam , Goldstein Lawrence S.B. 
Published: February 2009

AbstractThe motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIFl, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus‐directed microtubule movement up to 2 μm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.
Helix‐bundle membrane protein fold templates
Authors: Bowie James U. 
Published: February 2009

AbstractIn the fold recognition approach to structure prediction, a sequence is tested for compatibility with an already known fold. For membrane proteins, however, few folds have been determined experimentally. Here the feasibility of computing the vast majority of likely membrane protein folds is tested. The results indicate that conformation space can be effectively sampled for small numbers of helices. The vast majority of potential monomeric membrane protein structures can be represented by about 30‐folds for three helices, but increases exponentially to about 1, 500, 000 folds for seven helices. The generated folds could serve as templates for fold recognition or as starting points for conformational searches that are well distributed throughout conformation space.
Protein structure determination using a database of interatomic distance probabilities
Authors: Wall Michael E. , Phillips George N. , Subramaniam Shankar 
Published: February 2009

AbstractThe accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X‐ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side‐chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side‐chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high‐resolution refinements were improved, as is shown by an R‐factor analysis. The improvements were greatest when refinements were carried out using X‐ray data truncated at 3.5 Å. The database potential should therefore be a valuable tool for determining X‐ray structures, especially when only low‐resolution data are available.
Influence of key residues on the reaction mechanism of the cAMP‐dependent protein kinase
Authors: Hutter Michael C. , Helms Volkhard 
Published: February 2009

AbstractThe reaction mechanism of the catalytic phosphoryl transfer of cAMP‐dependent protein kinase (cAPK) was investigated by semi‐empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol−1 and the reaction itself to be exothermic by 12.2 kcal mol−1. The active site residue Asp 166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two‐dimensional energy hypersurfaces of the protein kinase wild‐type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys 168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.
Comparison of the kinetics of S‐S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme
Authors: Roux Pascale , Chaffotte Alain‐François , Goldberg Michel E. , Ruoppolo Margherita 
Published: February 2009

AbstractTo investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray‐mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far‐UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.
Effect of hydrostatic pressure on unfolding of α‐lactalbumin: Volumetric equivalence of the molten globule and unfolded state
Authors: Kobashigawa Yoshihiro , Nitta Katsutoshi , Sakurai Masao 
Published: February 2009

AbstractThe effect of pressure on the unfolding of bovine α‐lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, ΔV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The ΔV was estimated to be −63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that ΔV was −66 cm3/mol at 25 °C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of α‐lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.
Mass spectral characterization of a protein—nucleic acid photocrosslink
Authors: Golden Mace C. , Resing Katheryn A. , Koch Tad H. , Collins Brian D. , Willis Michael C. 
Published: February 2009

AbstractA photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61‐mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross‐linked protein‐nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130–138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.
Pressure‐induced thermostabilization of glutamate dehydrogenase from the hyperthermophile pyrococcus furiosus
Authors: Sun MICHAEL M.C. , Tolliday Nicola , Vetriani Costantino , Robb Frank T. , Clark Douglas S. 
Published: February 2009

AbstractIn this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophilePyrococcus furiosus(Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C‐terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin‐label attached to the terminal cysteine of rGDHtrevealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion‐pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36‐fold for rGDHtat 105 C and 750 atm, the largest pressure‐induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHtwas also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure‐induced thermostabilization is proposed.
The calorimetric criterion for a two‐state process revisited
Authors: Zhou Yaoqi , Hall Carol K. , Karplus Martin 
Published: February 2009

AbstractThe “calorimetric criterion” is one of the important experimental approaches for determining whether protein folding is an “all‐or‐none” two‐state transition (i.e., whether intermediates are present at equilibrium). The calorimetric criterion states that the equivalence of the “measured” calorimetric enthalpy change and the effective two‐state van't Hoff enthalpy change demonstrates that there is a two‐state transition. This paper addresses the essential question of whether the calorimetric criterion is a necessary and sufficient condition for a two‐state process and shows that it is necessary but not sufficient by means of specific examples. Analysis of simple models indicates that the heat capacity curve, regardless of whether it originates from a two‐state process or not, can always be decomposed in such a way that the calorimetric criterion is satisfied. Exact results for a three‐state model and a homopolymer tetramer demonstrate that the deviation from the calorimetric criterion is not simply related to the population of intermediate states. Analysis of a three‐helix bundle protein model, which has a two‐state folding from a random coil to ordered (molten) globule, shows that the calorimetric criterion may not be satisfied if the standard linear interpolation of baselines (weighted or unweighted) is employed. A specific example also suggests that the more recently introduced deconvolution method is not necessarily better than the simple calorimetric criterion for distinguishing a two‐state transition from a three‐state transition. Although the calorimetric criterion is not a sufficient condition for a two‐state process, it is likely to continue to be of practical utility, particularly when its results are shown to be consistent with those from other experimental methods.
Structural bases of lectin‐carbohydrate affinities: Comparison with protein‐folding energetics
Authors: García‐Hernández Enrique , Hernández‐Arana Andrés 
Published: February 2009

AbstractWe have made a comparative structure based analysis of the thermodynamics of lectin‐carbohydrate (L‐C) binding and protein folding. Examination of the total change in accessible surface area in those processes revealed a much larger decrease in free energy per unit of area buried in the case of L‐C associations. According to our analysis, this larger stabilization of L‐C interactions arises from a more favorable enthalpy of burying a unit of polar surface area, and from higher proportions of polar areas. Hydrogen bonds present at 14 L‐C interfaces were identified, and their overall characteristics were compared to those reported before for hydrogen bonds in protein structures. Three major factors might explain why polar–polar interactions are stronger in L‐C binding than in protein folding: (1) higher surface density of hydrogen bonds; (2) better hydrogen‐bonding geometry; (3) larger proportion of hydrogen bonds involving charged groups. Theoretically, the binding entropy can be partitioned into three main contributions: entropy changes due to surface desolvation, entropy losses arising from freezing rotatable bonds, and entropic effects that result from restricting translation and overall rotation motions. These contributions were estimated from structural information and added up to give calculated binding entropies. Good correlation between experimental and calculated values was observed when solvation effects were treated according to a parametrization developed by other authors from protein folding studies. Finally, our structural parametrization gave calculated free energies that deviate from experimental values by 1.1 kcal/mol on the average; this amounts to an uncertainty of one order of magnitude in the binding constant.
Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates
Authors: D'Silva Patrick R. , Lala Anil K. 
Published: February 2009

AbstractRecent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent—2[3H]diazofluorene. This carbene‐based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels α‐lactalbumin as a function of pH (3–7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23–34) and C (86–98) helix of the native structure are extensively labeled. The small β‐domain (40–50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of α‐lactalbumin, the α‐domain has a greater degree of persistent structure than the β‐domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.
From fold predictions to function predictions: Automation of functional site conservation analysis for functional genome predictions
Authors: Zhang Baohong , Rychlewski Leszek , Pawlowski Krzysztof , Fetrow Jacquelyn S. , Skolnick Jeffrey , Godzik Adam 
Published: February 2009

AbstractA database of functional sites for proteins with known structures, SITE, is constructed and used in conjunction with a simple pattern matching program SiteMatch to evaluate possible function conservation in a recently constructed database of fold predictions for Escherichia coli proteins (Rychlewski L et al., 1999, Protein Sci 8:614–624). In this and other prediction databases, fold predictions are based on algorithms that can recognize weak sequence similarities and putatively assign new proteins into already characterized protein families. It is not clear whether such sequence similarities arise from distant homologies or general similarity of physicochemical features along the sequence. Leaving aside the important question of nature of relations within fold superfamilies, it is possible to assess possible function conservation by looking at the pattern of conservation of crucial functional residues. SITE consists of a multilevel function description based on structure annotations and structure analyses. In particular, active site residues, ligand binding residues, and patterns of hydrophobic residues on the protein surface are used to describe different functional features. SiteMatch, a simple pattern matching program, is designed to check the conservation of residues involved in protein activity in alignments generated by any alignment method. Here, this procedure is used to study conservation of functional features in alignments between protein sequences from the E. coli genome and their optimal structural templates. The optimal templates were identified and alignments taken from the database of genomic structural predictions was described in a previous publication (Rychlewski L et al., 1999, Protein Sci 8:614–624). An automated assessment of function conservation is used to analyze the relation between fold and function similarity for a large number of fold predictions. For instance, it is shown that identifying low significance predictions with a high level of functional residue conservations can be used to extend the prediction sensitivity for fold prediction methods. Over 100 new fold/function predictions in this class were obtained in the E. coli genome. At the same time, about 30% of our previous fold predictions are not confirmed as function predictions, further highlighting the problem of function divergence in fold superfamilies.