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PubMed2000

Analysis of aluminum-yeast hexokinase interaction: modifications on protein structure and functionality.

The aluminum and yeast hexokinase interaction was studied. Structural changes were correlated with variations in protein functionality. Results show two different behaviors: At low metal concentrations preferential adsorption of metal (and water exclusion) induces aggregate formation. No significant changes in the protein structure occur, but there is a continuous loss of activity (from the first concentration). At large salt concentrations a monomerization process and a conformational change in the secondary structure as well as in the three-dimensional structure take place. This change reduces the percentage of alpha-helix conformation, gives thermal stability to the protein, and allows the exposure of some tryptophan residue and hydrophobic regions. The protein inhibition increases. Conformational change and monomerization may allow access of the metal to the substrate site, mainly the ATP site. The inhibition in any case is of mixed type with a competitive component.

Adenosine Triphosphate↗
PubMed1984

Ribosome conformational changes associated with protein S6 phosphorylation.

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using ribosomes with unphosphorylated, and extensively phosphorylated S6. Comparison of the results indicated that proteins S3, S4, S7, and S23/24 of the small subunit, and proteins L9, L10, L12, L18, L27, L34, and L36 are involved in a ribosomal conformational change.

Animals↗
PubMed1995

Effects of hydrostatic pressure on the kinetics reveal a volume increase during the bacteriorhodopsin photocycle.

A protein structural change in the photocycle of the proton pump, bacteriorhodopsin, detected earlier in the M photointermediate by diffraction, consists mainly of changes at the cytoplasmic surface that include an outward tilt of the cytoplasmic end of helix F. Such a conformational rearrangement would result in greater exposure of the interhelical cavity to the medium, increased binding of water, and thus an increase in volume. In order to correlate the structural change with the kinetics of the photoreaction cycle, we measured the effects of hydrostatic pressure between 1 bar and 1 kbar on the rate constants of the photocycles of wild type bacteriorhodopsin and the D96N mutant. Combining the results provided all of the activation volumes and, therefore, the changes of volume in the various states after the K photointermediate is formed. There is an approximately 32 mL/mol volume increase after deprotonation of the retinal Schiff base to the extracellular side, during the M1 --> M2 reaction, that is not reversed until well after its reprotonation from the cytoplasmic side. The magnitude of this volume increase is about as predicted by the increase of the lattice constant in the M state. It occurs in the photocycle at the proposed reprotonation switch, supporting the idea that this conformation change is what alters the accessibility of the Schiff base from one membrane side to the other. Additionally, we observe a large positive (approximately 50 mL/mol) activation volume for proton exchange between D96 and the Schiff base of the wild type protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed2000

Benzodiazepines induce a conformational change in the region of the gamma-aminobutyric acid type A receptor alpha(1)-subunit M3 membrane-spanning segment.

Benzodiazepine binding to gamma-aminobutyric acid type A (GABA(A)) receptors allosterically modulates GABA binding and increases the currents induced by submaximal GABA concentrations. Benzodiazepines induce conformational changes in the GABA-binding site in the extracellular domain, but it is uncertain whether these conformational changes extend into the membrane-spanning domain where the channel gate is located. Alone, benzodiazepines do not open the channel. We used the substituted-cysteine-accessibility method to investigate diazepam-induced conformational changes in the region of the alpha(1)-subunit M3 membrane-spanning segment. In the absence of diazepam or GABA, pCMBS(-) did not react at a measurable rate with cysteine-substitution mutants between alpha(1)Phe296 and alpha(1)Glu303. In the presence of 100 nM diazepam, pCMBS(-) reacted with alpha(1)F296C, alpha(1)F298C, and alpha(1)L301C but not with the other cysteine mutants between alpha(1)Phe296 and alpha(1)Glu303. These three mutants are a subset of the five residues that we previously showed reacted with pCMBS(-) applied in the presence of GABA. The pCMBS(-) reaction rates with these three cysteine mutants were similar in the presence of diazepam and GABA. Thus, diazepam, which binds to the extracellular domain, induces a conformational change in the membrane-spanning domain that is similar to a portion of the change induced by GABA. Because diazepam does not open the channel, these results provide structural evidence that the diazepam-bound state represents an intermediate conformation distinct from the open and resting/closed states of the receptor. The diazepam-induced conformational change in the M3 segment vicinity may be related to the mechanism of allosteric potentiation.

4-Chloromercuribenzenesulfonate↗
PubMed1990

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.

Aspergillus oryzae↗
PubMed2000

Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites.

We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.

DNA↗
PubMed1986

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes.

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.

Circular Dichroism↗
PubMed1990

Conformational analysis of inulobiose by molecular mechanics.

Conformational energies for inulobiose [beta-D-fructofuranosyl-(2----1)-beta-D-fructofuranoside], a model for inulin, were computed with the molecular mechanics program MMP2(85). The torsion angles of the three linkage bonds were driven in 20 degree increments, and the steric energy of all other parameters was minimized. The linkage torsion angles defined by C-1'-C-2'-O-C-1 (phi) and O-C-1-C-2-O-2 (omega) have minima at +60 degrees and -60 degrees, respectively, regardless of side group orientation; accessible minima exist at other staggered conformations. The torsion angle at the central bond C-2'-O-1-C-1-C-2 (psi) was approximately 180 degrees in all the low-energy conformers. This appears to be generally true for rings linked by three bonds. The fructofuranose rings initially had low-energy 4/3T conformations (angle of pseudorotation, phi 2 = 265 degrees) that were retained except when the linkage conformations created severe inter-residue conflicts. In those cases, almost all puckerings of the furanose rings were found.

Carbohydrate Conformation↗
PubMed1998

Conformational energy penalties of protein-bound ligands.

The conformational energies required for ligands to adopt their bioactive conformations were calculated for 33 ligand-protein complexes including 28 different ligands. In order to monitor the force field dependence of the results, two force fields, MM3* and AMBER*, were employed for the calculations. Conformational analyses were performed in vacuo and in aqueous solution by using the generalized Born/solvent accessible surface (GB/SA) solvation model. The protein-bound conformations were relaxed by using flat-bottomed Cartesian constraints. For about 70% of the ligand-protein complexes studied, the conformational energies of the bioactive conformations were calculated to be < or = 3 kcal/mol. It is demonstrated that the aqueous conformational ensemble for the unbound ligand must be used as a reference state in this type of calculations. The calculations for the ligand-protein complexes with conformational energy penalties of the ligand calculated to be larger than 3 kcal/mol suffer from uncertainties in the interpretation of the experimental data or limitations of the computational methods. For example, in the case of long-chain flexible ligands (e.g. fatty acids), it is demonstrated that several conformations may be found which are very similar to the conformation determined by X-ray crystallography and which display significantly lower conformational energy penalties for binding than obtained by using the experimental conformation. For strongly polar molecules, e.g. amino acids, the results indicate that further developments of the force fields and of the dielectric continuum solvation model are required for reliable calculations on the conformational properties of this type of compounds.

Binding Sites↗
PubMed1995

Processing of the precursors to neurotensin and other bioactive peptides by cathepsin E.

Cathepsin E (EC 3.4.23.34), an intracellular aspartic proteinase, was purified from monkey intestine by simple procedures that included affinity chromatography and fast protein liquid chromatography. Cathepsin E was very active at weakly acidic pH in the processing of chemically synthesized precursors such as the precursor to neurotensin/neuromedin, proopiomelanocortin, the precursor to xenopsin, and angiotensinogen. The processing sites were adjacent to a dibasic motif in the former two precursors and at hydrophobic recognition sites in the latter two. The common structural features that specified the processing sites were found in the carboxyl-terminal sequences of the active peptide moieties of these precursors; namely, the sequence Pro-Xaa-X'aa-hydrophobic amino acid was found at positions P4 through P1. Pro at the P4 position is thought to be important for directing the processing sites of the various precursor molecules to the active site of cathepsin E. Although the positions of Xaa and X'aa were occupied by various amino acids, including hydrophobic and aromatic amino acids, some of these had a negative effect, as typically observed when Glu/Arg and Pro were present at the P3 and P2 positions, respectively. Cathepsin D was much less active or was almost inactive in the processing of the precursors to neurotensin and related peptides as a result of the inability of the Pro-directed conformation of the precursor molecules to gain access to the active site of cathepsin D. Thus, the consensus sequence of precursors, Pro-Xaa-X'aa-hydrophobic amino acid, might not only generate the best conformation for cleavage by cathepsin E but might be responsible for the difference in specificities between cathepsins E and D.

Amino Acid Sequence↗
PubMed1997

Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.

Bacillus subtilis↗
PubMed1992

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 2. Denaturation and structural stability.

The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis. Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding. The mechanism by which the protein achieves thermal stabilization is quite unusual. It is driven by a conformational equilibrium between two forms of different stability. The stability of each form towards denaturation is characterized by a specific temperature dependence. The low-temperature form, indicated as 'form A', is stable over 12-89 degrees C. Its stability maximum is 36.8 kJ/mol at 50 degrees C. 'Form B', which is populated at higher temperature, spans the interval 28-146 degrees C. Its stability maximum is 71.6 kJ/mol at 87 degrees C. A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e. hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy. The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding. In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability.

Enzyme Stability↗
PubMed1999

Mutant huntingtin forms in vivo complexes with distinct context-dependent conformations of the polyglutamine segment.

Huntington's disease (HD) is caused by an expanded glutamine tract, which confers a novel aggregation-promoting property on the 350-kDa huntingtin protein. Using specific antibodies, we have probed the structure of the polyglutamine segment in mutant huntingtin complexes formed in cell culture from either truncated or full-length protein. Complexes formed by a mutant amino terminal fragment most frequently entail a change in conformation that eliminates reactivity with the polyglutamine-specific mAb 1F8, coincident with production of insoluble aggregate. By contrast, complexes formed by the full-length mutant protein remain soluble and are invariably 1F8-reactive, indicating a soluble polyglutamine conformation. Therefore, aggregates in HD may form by different biochemical mechanisms that invoke different possibilities for the pathogenic process. If pathogenesis is triggered by a truncated fragment, it probably involves the formation of an insoluble aggregate. However, the observation of soluble complexes in which an HD-specific pathogenic conformation of the glutamine tract remains accessible suggests that pathogenesis could also be triggered at the level of full-length huntingtin by abnormal aggregation with normal or abnormal protein partners.

Animals↗
PubMed1995

Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops.

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Base Sequence↗
PubMed2001

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair.

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

Arginine↗
PubMed2000

Effect of limited proteolysis on phospholipase C-gamma1 kinetics.

Phospholipase C-gamma1 is a tightly regulated, multidomain protein that generates the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Kinetic analysis reveals that phospholipase C-gamma1 displays apparent allosteric behavior. A previous study determined that proteolytic cleavage of the SH domain region of phospholipase C-gamma1 yields an activated form of the enzyme (A. W. Fernald, G. A. Jones, and G. Carpenter Biochem. J. 302, 508, 1994). In this study, we show that activation of phospholipase C-gamma1 by proteolysis decreases both the cooperativity and the half-maximal value of the enzyme for substrate. Kinetic analysis revealed that the mole fraction of phosphatidylinositol 4,5-bisphosphate (PIP(2)) that resulted in half-maximal PIP(2) hydrolysis (S(0.5)) was lower for proteolyzed than uncleaved phospholipase C-gamma1 (0.08 mole fraction vs 0.18 mole fraction of PIP(2)). The cooperativity index was lower for proteolyzed than full-length phospholipase C-gamma1 (n = 2.5 vs n = 4). Kinetic analysis also revealed that the estimated dissociation constant was lower for phospholipase C-gamma1 that had been subjected to proteolysis (0.1 mM vs 1.0 mM PIP(2) for cleaved vs uncleaved phospholipase C-gamma1, respectively). It was previously hypothesized that activation of phospholipase C-gamma1 requires a conformational change that results in increased accessibility of substrate to the active site and that the SH domain of the enzyme is involved in the activation event. These experiments support the hypothesis that a portion of the protein covers the active site, allosterically inhibiting the enzyme, and that the removal of this "lid" domain activates the enzyme.

Allosteric Regulation↗
PubMed1991

Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

Actins↗
PubMed2005

Solvent constraints on the property space of acetylcholine. 2. Ordered media.

The objective of this study was to investigate the conformational and property spaces of acetylcholine in hydrated octanol and in a membrane model. Molecular dynamics simulations of long duration (15 ns) were carried out, yielding 3000 conformers. For each, we calculated N(+)-C8 distance, solvent-accessible surface area (SAS), polar surface area (PSA), dipole moment, and lipophilicity (virtual logP). Their variations as a function of the dihedral angles tau(2) and tau(3) remained unexpectedly broad and comparable to those seen previously in a vacuum, in water, and in chloroform.(12) Thus, each of the seven conformational clusters was able to access a marked proportion of the lipophilicity space accessible to acetylcholine (0.40 in the logP scale). Histograms of logP distributions revealed two overlapping populations, namely more lipophilic and more hydrophilic. Their deconvolution into two Gaussian curves demonstrated solvent-mediated constraints on the lipophilicity space of acetylcholine, clearly showing how a polar medium favors polar conformers, whereas the opposite is true for media of low polarity.

Acetylcholine↗