bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein conformer selection by ligand binding observed with crystallography
Authors: Cao Yi , Musah Rabi A. , Wilcox Sheri K. , Goodin David B. , McRee Duncan E. 
Published: July 2010

AbstractA large‐scale movement between “closed” and “open” conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of Trp191 in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop Pro190‐Asn195 and exposing Trp191 to the protein surface. Kinetic measurements are consistent with a two‐step binding mechanism in which the rate‐limiting step is a transition of the protein to the open state, which then binds the ligand. This large‐scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild‐type protein is in the open form in solution at any given time.
Prime region subsite specificity characterization of human cathepsin D: The dominant role of position 128
Authors: Beyer Brian M. , Dunn Ben M. 
Published: July 2010

AbstractIn order to contribute to our understanding of cathepsin D (CatD) active site specificity, two series of chromogenic octapeptides with systematic substitutions in positions P'2 and P'3 were synthesized. This panel was characterized with native human liver cathepsin D (nHuCatD) and yielded information concerning specificity trends within the S'2 and S'3 subsites. The pepstatin inhibited crystal structure of nHuCatD (Baldwin et al., 1993) was then utilized in conjunction with these subsite preference data to identify residues suspected of contributing to “prime” side subsite specificity. These residues were targeted for site‐directed mutagenesis using the re‐engineered recombinant model, “short” pseudocathepsin D (Beyer & Dunn, 1996). As a result of these analyses it was determined that prime region subsites do contribute to the unique specificity of human CatD. Furthermore, it was ascertained that the poly‐proline loop does not have an active role in S'3 subsite specificity. Lastly, it appears that Ile 128 has a dominant role on S'2 subsite specificity whereas Val130 does not.
NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family
Authors: Liu Jianhua , Gong Yuxi , Prakash Om , Krishnamoorthi Ramaswamy , Wen Lisa , Lee Insuk , Huang Jenq‐Kuen 
Published: July 2010

AbstractSerine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49593‐626), whereby an enzyme‐catalyzed equilibrium between intact (I) and reactive‐site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI‐III; Mr 3 kDa) of the squash family and rCMTI‐V (Mr ∼ 7 kDa) of the potato I family. These two inhibitors have different binding loop‐scaffold interactions and different Khyd values—2.4 (CMTI‐III) and 9 (CMTI‐V)—at 25°C. The reactive‐site peptide bond (P1‐P'1) is that between Arg5 and Ile6 in CMTI‐III, and that between Lys44 and Asp45 in CMTI‐V. The order parameters (S2) of backbone NHs of uniformly 15N‐labeled rCMTI‐III and rCMTI‐III* were determined from measurements of 15N spin‐lattice and spin‐spin relaxation rates, and {1H}‐15N steady‐state heteronuclear Overhauser effects, using the model‐free formalism, and compared with the data reported previously for rCMTI‐V and rCMTI‐V*. The backbones of rCMTI‐III (〈S2〉 = 0.71) and rCMTI‐III* (〈S2) = 0.63) are more flexible than those of rCMTI‐V (〈S2〉 = 0.83) and rCMTI‐V* (〈S2) = 0.85). The binding loop residues, P4‐P1, in the two proteins show the following average order parameters: 0.57 (rCMTI‐III) and 0.44 (rCMTI‐III*); 0.70 (rCMTI‐V) and 0.40 (rCMTI‐V*). The P1′‐P4′ residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI‐III) and 0.47 (rCMTI‐III*); and 0.73 (rCMTI‐V) and 0.83 (rCMTI‐V*). The newly formed C‐terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI‐III* due to the Cys3‐Cys20 crosslink. In contrast, the newly formed N‐terminal (Pn′ residues) becomes more flexible only in rCMTI‐III*, most likely due to lack of an interaction between the P1′ residue and the scaffold in rCMTI‐III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn′ residues seem to facilitate the resynthesis of the P1‐P1′ bond, leading to a lower Khyd value.
The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect
Authors: Xu Jian , Baase Walter A. , Baldwin Enoch , Matthews Brian W. 
Published: July 2010

AbstractTo further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non‐polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the “large‐to‐small” substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts ≤ 0.3 Å); in other cases mainchain atoms moved up to 2 Å. In the case of Ile 29 → Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 → Ala (V149A) and Met 6 → Ala (M6A), however, are exceptions and have, respectively, one and two well‐ordered water molecules within the cavity. The Val 149 → Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild‐type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 → Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., − 2.1 kcal/mol for all Leu → Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol−1 Å−3, Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu → Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178‐183).
Fragment of GABAAreceptor containing key ligand‐binding residues overexpressed inEscherichia coli
Authors: Xue Hong , Chu Ruiai , Hang Jun , Lee Peggy , Zheng Hui 
Published: July 2010

AbstractGABAAreceptor plays a major role in inhibitory synaptic transmission in the central nervous system and is the target of drugs such as the benzodiazepine tranquilizers. The polymeric membrane protein nature of GABAAreceptor has rendered structural elucidation of the receptor a formidable task, greatly hampering structure‐based drug design. We report here the first expression inEscherichia coliof a fragment of GABAAreceptor. This 131‐ residue fragment, spanning Cys166 to Leu296 of human GABAAreceptor α1 subunit, contains residues previously suggested to be involved in benzodiazepine binding. The overexpressed nonfusion recombinant protein was purified to near homogeneity and characterized by circular dichroism (CD), which showed that the recombinant protein has well defined secondary structures where β‐strands are dominant. The stability of the secondary structures was demonstrated by CD spectra at high pH and elevated temperature. Excluding part of the sequences from the carboxyl terminal of the fragment resulted in dramatic changes in the secondary structures comparable to the effects caused by SDS denaturation. Our results therefore suggest that the 131‐residue fragment harbors an integral structural domain of the receptor. The overexpression of the recombinant protein fragment thus opens the way to the biochemical and structural studies of a functionally important region of the receptor, and exemplifies an effective approach of expression and characterization that potentially may be extended to other members of the ligand gated channel receptor superfamily, to which the GABAAreceptor belongs.
Domain assignment for protein structures using a consensus approach: Characterization and analysis
Authors: Jones Susan , Stewart Michael , Michie Alex , Swindells Mark B. , Orengo Chirstine , Thornton Janet M. 
Published: July 2010

AbstractA consensus approach for the assignment of structural domains in proteins is presented. The approach combines a number of previously published algorithms, and takes advantage of the elevated accuracy obtained when assignments from the individual algorithms are in agreement. The consensus approach is tested on a data set of 55 protein chains, for which domain assignments from four automated methods were known, and for which crystallographers assignments had been reported in the literature. Accuracy was found to increase in this test from 72% using individual algorithms to 100% when all four methods were in agreement. However a consensus prediction using all four methods was only possible for 52% of the dataset. The consensus approach (using three publicly available domain assignment algorithms (PUU, DETECTIVE, DOMAK)) was then used to make domain assignments for a data set of 787 protein chains from the Protein Data Bank. Analysis of the assignments showed 55.7% of assignments could be made automatically and of these, 13.5% were multi‐domain proteins. Of the remaining 44.3% that could not be assigned by the consensus procedure 90.4% had their domain boundaries assigned correctly by at least one of the algorithms. Once identified, these domains were analyzed for trends in their size and secondary structure class. In addition, the discontinuity of each domain along the protein chain was considered.
An interaction‐based analysis of calcium‐induced conformational changes in Ca2+ sensor proteins
Authors: Nelson Melanie R. , Chazin Walter J. 
Published: July 2010

AbstractCalcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding‐induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium‐induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF‐hands. A secondary analysis of the conformation of the C‐terminal domin of CaM (CaM‐C) clearly shows that CaM‐C occupies a closed conformation in the absence of calcium that is distinct from the semi‐open conformation observed in the C‐terminal EF‐hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF‐hand calcium‐binding protein family. Factors contributing to the stability of the Ca2+‐loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in “non‐sensor” proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.
Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor α convertase inhibitors
Authors: Xavier Gomis‐Rüth F. , Meyer Edgar F. , Kress Lawrence F. , Politi Vincenzo 
Published: July 2010

AbstractCrotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor α convertase, TACE, involved in the release of the inflammatory cytokine, TNFα. The structure of adamalysin II in noncovalent complex with two small‐molecule right‐hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X‐ray diffraction data up to 2.6 and 2.8 Å resolution. The inhibitors bind to the ‐side of the proteinase, inserting between two protein segments, establishing a mixed parallel‐antiparallel three‐stranded β‐sheet and coordinate the central zinc ion in a bidentate manner via their two C‐terminal oxygen atoms. The proteinase‐inhibitor complexes are described in detail and are compared with other known structures. An adamalysin‐based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.
Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1
Authors: Myers Jeffrey K. , Nick Pace C. , Martin Scholtz J. 
Published: July 2010

AbstractTrifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the α‐helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine‐based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.
The early folding kinetics of apomyoglobin
Authors: Pappu Rohit V. , Weaver David L. 
Published: July 2010

AbstractThe folding pathway of apomyoglobin has been experimentally shown to have early kinetic intermediates involving the A, B, G, and H helices. The earliest detected kinetic events occur on a ns to μis time scale. We show that the early folding kinetics of apomyoglobin may be understood as the association of nascent helices through a network of diffusioncollision‐coalescence steps G + H GH + A AGH + B ABGH obtained by solving the diffusion‐collision model in a chemical kinetics approximation. Our reproduction of the experimental results indicates that the model is a useful way to analyze folding data. One prediction from our fit is that the nascent A and H helices should be relatively more helix‐like before coalescence than the other apomyoglobin helices.
High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae al homeodomain expressed in Escherichia coli
Authors: Forman Michael D. , Stack Robert F. , Masters Paul S. , Hauer Charles R. , Baxter Susan M. 
Published: July 2010

AbstractThe Saccharomyces cerevisiae al homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared al homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5′‐AGA‐3′ sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in al homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5′‐AGA‐3′ tRNAARG, was co‐expressed in E. coli with the al‐encoding plasmid to produce a homogeneous recombinant al homeodomain. Co‐expression of the dnaY gene completely blocks mistranslation of arginine to lysine during al overexpression in minimal media, and homogeneous protein is produced.
A LexA mutant repressor with a relaxed inter‐domain linker
Authors: Oertel‐Buchheit Pascale , Reinbolt Joseph , Johan Matthias , Granger‐Schnarr Michèle , Schnarr Manfred 
Published: July 2010

AbstractThe LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino‐terminal DNA binding domain and a carboxy‐terminal dimerization domain. These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained. So far, no structure of any of the full‐length repressors is available. Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu‐C cleavage over a segment of at least 20 amino acids, whereas the LexA wild‐type hinge region is resistant to these proteases. These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter‐domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy‐terminal domain and/or a fairly stable local structure of the linker region.
Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles 
Published: July 2010

AbstractAlteromonas haloplanctis is a bacterium that flourishes in Antarctic sea‐water and it is considered as an extreme psychrophile. We have determined the crystal structures of the α‐amylase (AHA) secreted by this bacterium, in its native state to 2.0 Å resolution as well as in complex with Tris to 1.85 Å resolution. The structure of AHA, which is the first experimentally determined three‐dimensional structure of a psychrophilic enzyme, resembles those of other known α‐amylases of various origins with a surprisingly greatest similarity to mammalian α‐amylases. AHA contains a chlorideion which activates the hydrolytic cleavage of substrate α‐l,4‐glycosidic bonds. The chloride binding site is situated ∼5 Å from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation.A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine‐protease like catalytic triads was found ∼22 Å from the active site. We found that this triad is equally present in other chloride dependent α‐amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted α‐amylase.
Solvation studies of DMP323 and A76928 bound to HIV protease: Analysis of water sites using grand canonical Monte Carlo simulations
Authors: Marrone Tami J. , McCammon J. Andrew , Resat Haluk , Hodge C. Nicholas , Chang Chong‐Hwan 
Published: July 2010

AbstractWe examine the water solvation of the complex of the inhibitors DMP323 and A76928 bound to HIV‐1 protease through grand canonical Monte Carlo simulations, and demonstrate the ability of this method to reproduce crystal waters and effectively predict water positions not seen in the DMP323 or A76928 structures. The simulation method is useful for identifying structurally important waters that may not be resolved in the crystal structures. It can also be used to identify water positions around a putative drug candidate docked into a binding pocket. Knowledge of these water positions may be useful in designing drugs to utilize them as bridging groups or displace them in the binding pocket. In addition, the method should be useful in finding water sites in homology models of enzymes for which crystal structures are unavailable.
Solution structure of Compstatin, a potent complement inhibitor
Authors: Morikis Dimitrios , Assa‐Munt Nuria , Sahu Arvind , Lambris John D. 
Published: July 2010

AbstractThe third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13‐residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure‐function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry‐restrained simulated‐annealing methodology, using distance, dihedral angle, and 3JNH‐Hα‐coupling constant restraints. The Compstatin structure contained a type I β‐turn comprising the segment Gln5‐Asp6‐Trp7‐Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3JNH‐Hα‐coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100‐fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side‐chain interactions and the β‐turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.