Home About us Contact
|
Home About us Contact | ||
|
|
||
Open Conformation (open + conformation)
Selected AbstractsConformational effects on the performance and selectivity of a polymeric pseudostationary phase in electrokinetic chromatographyELECTROPHORESIS, Issue 4-5 2005Jonathan P. McCarney Abstract The effect of the conformation of a polymeric pseudostationary phase on performance and selectivity in electrokinetic chromatography was studied using an amphiphilic pH-responsive polymer that forms compact intramolecular aggregates (unimer micelles) at low pH and a more open conformation at high pH. The change in conformation was found to affect the electrophoretic mobility, retention, selectivity, and separation efficiency. The low-pH conformer has higher electrophoretic mobility and greater affinity for most solutes. The unimer micelle conformation was also found to provide a solvation environment more like that of micelles and other amphiphilic self-associative polymers studied previously. It was not possible to fully characterize the effect of conformation on efficiency, but very hydrophobic solutes with long alkyl chains appeared to migrate with better efficiency when the unimer micelle conformation was employed. The results imply that polymers with a carefully optimized lipophilic-hydrophilic balance that allow self-association will perform better as pseudostationary phases. In addition, the results show that electrokinetic chromatography is a useful method for determining the changes in solvation environment provided by stimuli-responsive polymers with changes in the conditions. [source] High resolution structure and catalysis of O -acetylserine sulfhydrylase isozyme B from Escherichia coliFEBS JOURNAL, Issue 20 2007Georg Zocher The crystal structure of the dimeric O -acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O -acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O -acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction. [source] Actin-binding domain of mouse plectinFEBS JOURNAL, Issue 10 2004Crystal structure, binding to vimentin Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2,) from mouse, to a resolution of 1.95 and 2.0 Å. Comparison of pleABD/2, with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2, has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks. [source] Crystal structures of bovine ,-lactoglobulin in the orthorhombic space group C2221FEBS JOURNAL, Issue 2 2001Structural differences between genetic variants A, features of the Tanford transition The crystal structures of ,-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C2221 (lattice Y) by X-ray diffraction at 2.0 Å and 1.95 Å resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci.8, 75,83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue. [source] Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: A quantitative modelPROTEIN SCIENCE, Issue 6 2000Tanja Schulz Abstract The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(HN, , Oalc) between the histidine side chain and the alcohol oxygen increases. d(HN, , Oalc) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(HN, , Oalc) is>2.2 Å for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(HN, , Oalc) is less than 2.0 Å, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E< 100), d(HN, , Oalc) is around 2.1 Å. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols. [source] Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomeraseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Mikko Salin Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM. The A-TIM crystals were grown in the presence of citrate, which is bound in the active site of each of the two molecules in the asymmetric unit. In this complex, the active-site loops loop-6 and loop-7 adopt the closed conformation, similar to that observed in liganded wild-type TIM. Extensive crystal-soaking protocols have been developed to flush the bound citrate out of the active-site pocket of both molecules and the crystal structure shows that the unliganded open conformation of the A-TIM active site is the same as in unliganded wild-type TIM. It is also shown that sulfonate compounds corresponding to the transition-state analogue 2-phosphoglycolate bind in the active site, which has a closed conformation. It is also shown that the new binding pocket of A-TIM can bind 3-phosphoglycerate (3PGA; an analogue of a C4-sugar phosphate) and 4-phospho- d -erythronohydroxamic acid (4PEH; an analogue of a C5-sugar phosphate). Therefore, these studies have provided a rationale for starting directed-evolution experiments aimed at generating the catalytic properties of a C5-sugar phosphate isomerase on the A-TIM framework. [source] Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornataACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009Zuxu Chen The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5,Å away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25,Å from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP. [source] Structure-assisted discovery of an aminothiazole derivative as a lead molecule for inhibition of bacterial fatty-acid synthesisACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2007Günter Pappenberger Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by ,-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25,µM as determined by fluorescence titration. A 1.35,Å crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein,ligand interaction offers strategies for further optimization of this low-molecular-weight compound. [source] The atomic resolution structure of human aldose reductase reveals that rearrangement of a bound ligand allows the opening of the safety-belt loopACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2007Marianna Biadene The crystal structure of human aldose reductase in complex with citrate has been determined to a resolution of 0.82,Å. The difference electron density for H atoms unequivocally shows that the cofactor is in the oxidized state corresponding to the situation after the catalytic event has occurred. A citrate molecule bound to the active site has been modelled in two different conformations. These two conformations correlate with a fully closed and a partially open conformation of the so-called safety-belt loop (Gly213,Ser226). The open conformation is observed for the first time with the cofactor bound to the protein and may be related to the initial phase of the opening of the safety belt. The structure suggests that after the catalytic event, a rearrangement of a bound ligand can trigger the opening of the safety-belt loop, thus initiating the release of the oxidized cofactor. [source] Structural asymmetry and intersubunit communication in muscle creatine kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2007Jeffrey F. Ohren The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65,Å resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation. [source] The structure of Cryptococcus neoformans thymidylate synthase suggests strategies for using target dynamics for species-specific inhibitionACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2005Robert H. O'Neil The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1,Å structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS,dUMP,inhibitor complexes with a greater range of antifolates than human TS. 3,,3,,-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (Ki = 4,µM) over human TS (Ki >> 245,µM). The E. coli TS,dUMP,GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered [Phan et al. (2001), J. Biol. Chem.276, 14170,14177] may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery. [source] High-resolution structure of human phosphoserine phosphatase in open conformationACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2003Yves Peeraer The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53,Å. The crystals are orthorhombic, belonging to space group C2221, with unit-cell parameters a = 49.03, b = 130.25, c = 157.29,Å. The asymmetric unit contains two molecules. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (Rfree = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the l -serine biosynthesis pathway. It catalyses the Mg2+ -dependent hydrolysis of l -phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion. [source] The structural plasticity of Tom71 for mitochondrial precursor translocationsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Jingzhi Li Mitochondrial precursors are transported through the translocase of the outer membrane (TOM) complex. Tom70/Tom71 is a major surface receptor of the TOM complex for mitochondrial precursors and facilitates Hsp70/Hsp90-escorted precursor translocation into the mitochondrion. Previous structural studies of Tom71 have revealed that it contains an N-terminal and a C-terminal domain and that the two domains may remain in an open conformation when binding to Hsp70/Hsp90. In a newly obtained crystal form of a complex of Tom71 and the Hsp70 C-terminus, the N-terminal domain was found to have rotated about 12° towards the C-terminal domain compared with the previous determined crystal structure of Tom71 in the open conformation. This newly solved structure is defined as the `intermediate conformation'. The domain rearrangements in Tom71 significantly change the surface hydrophobicity and the volume of the precursor-binding pocket. This work suggests that Tom70/Tom71-family members may exhibit structural plasticity from the intermediate conformation to the fully open conformation when complexed with Hsp70/Hsp90. This structural plasticity enables the precursor receptors to accommodate different precursor substrates for mitochondrial translocation. [source] Structure of Escherichia coli malate dehydrogenase at 1.45,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Jelena Zaitseva The structure of apo malate dehydrogenase from Escherichia coli has been determined to 1.45,Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 146.0, b = 52.0, c = 168.9,Å, , = 102.2°. The structure was determined with the molecular-replacement pipeline program BALBES and was refined to a final R factor of 18.6% (Rfree = 21.4%). The final model has two dimers in the asymmetric unit. In each dimer one monomer contains the active-site loop in the open conformation, whereas in the opposing monomer the active-site loop is disordered. [source] Complexes of the copper-containing amine oxidase from Arthrobacter globiformis with the inhibitors benzylhydrazine and tranylcypromineACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2008David B. Langley Complexes of Arthrobacter globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65,Å resolution, respectively. Both inhibitors form covalent adducts with the TPQ cofactor. A tyrosine residue, proposed to act as a gate to the AGAO active site, is in its open conformation. [source] | ||||||||||