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At least 19 recordsLinked to original sources
PubMed1988

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin.

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.

Animals↗
PubMedDate not supplied

Statistical mechanical treatment of protein conformation. III. Prediction of protein conformation based on a three-state model.

The method proposed for the evaluation of statistical weights in paper I, and the three-state model [alpha-helical (alpha), extended (epsilon), and other (c) states] formulated in paper II, have been used to develop a procedure to predict the backbone conformations of proteins, based on the concept of the predominant role played by shortrange interactions in determining protein conformation. Conformational probability profiles, in which the probabilities of formation of three consecutive alpha-helical conformations (triad) and of four consecutive extended conformations (tetrad) have been defined relative to their average values over the whole molecule, are calculated for 19 proteins, of which 16 had been used in paper I to evaluate the set of statistical weights of the 20 naturally occurring amino acids. By comparing these conformational probability profiles to experimental x-ray observations, the following results have been obtained: 80% of the alpha-helical regions and 72% of the extended conformational regions have been predicted correctly for the 19 proteins. The percentage of residues predicted correctly is in the range of 53 to 90% for the alpha-helical conformation and in the range of 63 to 88% for the extended conformation for the 19 proteins in the two-state models [alpha-helical (alpha) and other (c) states, and extended (epsilon) and other (c) states]. In the three-state model, the percentage of residues predicted correctly is in the range of 47% to 77 for 19 proteins. These results suggest that the assumption of the dominance of short-range interactions, on which the predictive scheme is based, is a reasonable one. The present predictive method is compared with that of other authors.

Amino Acids↗
PubMed2006

Design of protein conformational switches.

Protein conformational switches are ubiquitous in nature and often regulate key biological processes. To design new proteins that can switch conformation, protein designers have focused on the two key components of protein switches: the amino acid sequence must be compatible with the multiple target states and there must be a mechanism for perturbing the relative stability of these states. Proteins have been designed that can switch between folded and disordered states, between distinct folded states and between different aggregation states. A variety of trigger mechanisms have been used, including pH shifts, post-translational modification and ligand binding. Recently, computational protein design methods have been applied to switch design. These include algorithms for designing novel ligand-binding sites and simultaneously optimizing a sequence for multiple target structures.

Protein Conformation↗
PubMedDate not supplied

Statistical mechanical treatment of protein conformation. 6. Elimination of empirical rules for prediction by use of a high-order probability. Correlation between the amino acid sequences and conformations for homologous neurotoxin proteins.

One-dimensional short-range interaction models for specific-sequence copolymers of amino acids have been developed in this series of papers. In this paper, a general method for predicting protein conformation (that is based on a one-dimensional short-range interaction model, and eliminates the need for the empirical rules introduced in papers III and IV) is described. The present method involves the use of conformational (or conformational-sequence) probabilities of higher order than the first- or second-order probabilities used in papers IV and V, i.e., it treats a sequence of any number of residues; it thus alters the predictive methods that involved empirical rules in papers III and IV, and low-order (first- or second-order) probabilities in papers IV and V. The general method is applied here to the prediction of the backbone conformations of proteins, using the three-state model [helical (h), extended (epilson), and other coil (c) states] proposed in the theoretical formulation of paper II. The statistical weights in the three-state model are evaluated from the atomic coordinates of the x-ray structures of 26 proteins. The conformational-sequence probabilities (taken for three consecutive residues for numerical computation in this paper) are calculated for all possible triads (i.e., for all possible combinations of the three states, h, epilson, and c for each residue) for bovine pancreatic trypsin inhibitor and clostridial flavodoxin, in order to select the most probable conformations of these proteins. The predicted results for these proteins are compared to those predicted in paper III and to those observed experimentally. The method is applied further to the prediction of the backbone structures of homologous neurotoxin proteins whose amino acid sequences are known but whose x-ray structures are not. The effects of variation in the amino acid sequence on the conformations of the backbones are discussed from the point of view of the homologies in the amino acid sequences of 19 neurotoxins. Application of the present general predictive method to a four- and a multistate model is also described.

Amino Acid Sequence↗
PubMed1978

The use of gel filtration to follow conformational changes in proteins. Conformational flexibility of bovine myelin basic protein.

The hydrodynamic behavior of bovine myelin basic protein was studied by gel filtration through Sephadex G-100 under conditions which included variations in pH from 2 to 12, variations in ionic strength from 0.01 to 1.5 M at pH 2 and from 0.1 to 2 M at pH 7, and variations in guanidinium chloride concentration from 0 to 6 M. A number of well characterized compact globular proteins were subjected to the same conditions for comparison. Compact globular proteins showed major conformational transitions due to acid, alkali, and guanidinium chloride denaturation and, possibly, minor transitions as well. Myelin basic protein behaved like a flexible linear polyelectrolyte, expanding continuously between pH 11 and pH 2 to 3 at ionic strength 0.1 M and contracting continuously with increase in ionic strength at pH 2 and at pH 7 to the point of salting-out. Relatively low concentrations of guanidinium chloride (approximately 0.5 M) were sufficient to cause the basic protein to expand. With increasing concentration of the denaturant the molecule continued to expand, but in a noncooperative manner. These results demonstrated the lack of significant intramolecular stabilization in the protein.

Animals↗
PubMed2005

Probing single-molecule protein conformational dynamics.

Protein conformational fluctuations and dynamics, often complex and associated with inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins are involved in multiple-step, multiple-conformation complex chemical interactions and transformations, such as in enzymatic reactions, protein-protein interactions, and ion-channel membrane protein processes. Alternatively, single-molecule spectroscopy is a powerful approach to probing and analyzing protein conformational dynamics in real time.

Bacteriophage T4↗
PubMed2005

Use of yeast as a model system to investigate protein conformational diseases.

Protein conformational diseases arise when a cellular protein adopts an aberrant shape that either directly or indirectly alters the physiology of its host cell. Notable conformational diseases include cystic fibrosis, Huntington's disease, the prion-related diseases, Alzheimer's disease, and antitrypsin deficiency. In principle, the severity and progression of conformational diseases can be altered by cellular factors that recognize and attempt to ameliorate the harmful effects of the disease-causing, misshapen protein. To better define the mechanistic underpinnings of cellular factors that mediate quality control, and to understand why a single misfolded protein can impact cell viability, specific proteins that cause each of the diseases listed above have been expressed in a model eukaryote, the yeast Saccharomyces cerevisiae. In this review, we describe what has been learned from these studies, and speculate on future uses of yeast expression systems.

Animals↗
PubMed2003

The androgen receptor transactivation domain: the interplay between protein conformation and protein-protein interactions.

The AR (androgen receptor) belongs to the nuclear receptor superfamily and directly regulates patterns of gene expression in response to the steroids testosterone and dihydrotestosterone. Sequences within the large N-terminal domain of the receptor have been shown to be important for transactivation and protein-protein interactions; however, little is known about the structure and folding of this region. Folding of the AR transactivation domain was observed in the presence of the helix-stabilizing solvent trifluorethanol and the natural osmolyte TMAO (trimethylamine N-oxide). TMAO resulted in the movement of two tryptophan residues to a less solvent-exposed environment and the formation of a protease-resistant conformation. Critically, binding to a target protein, the RAP74 subunit of the general transcription factor TFIIF, resulted in a similar resistance to protease digestion, consistent with induced folding of the receptor transactivation domain. Our current hypothesis is that the folding of the transactivation domain in response to specific protein-protein interactions creates a platform for subsequent interactions, resulting in the formation of a competent transcriptional activation complex.

Hormones↗
PubMed1999

Conformational spectra--probing protein conformational changes.

Stafford [Biophys. J. 17 (1996) MP452] has shown that it is possible, using the analytical ultracentrifuge in sedimentation velocity mode, to calculate the molecular weights of proteins with a precision of approximately 5%, by fitting Gaussian distributions to g(s*) profiles so long as partial specific volume and the radial position of the meniscus are known. This makes possible the analysis of systems containing several components by the fitting of multiple distributions to the total g(s*) profile. We have found the Stafford relationship to hold for a range of protein solutes, particularly good agreement being found when the g(s*) profiles are computed from Schlieren (dc/dr vs. r) data using the Bridgman equation [J. Am. Chem. Soc. 64 (1942) 2349] . On this basis, we have developed a new approach to the analysis of systems where two or more distinguishable conformations of a single species are present, either in the same sample cell or in different cells in the same rotor. In the former case, this allows us to analyse a given solution of pure protein (i.e. monodisperse with respect to M) to reveal the presence in that solution of two or more conformers under identical solvent conditions. In the latter case, we can detect with high sensitivity any conformational change occurring in the transition from one set of solvent conditions to another. Alternatively, in this case, we can analyse slightly different proteins (e.g. deletion mutants) for conformational changes under identical solvent conditions. Examples of these procedures using well-defined protein systems are given.

Journal Article↗
PubMed1983

ESR measurements on the effects of ethanol on the lipid and protein conformation in biological membranes.

The effects of ethanol on the conformation of proteins or fluidity of lipids was studied in human erythrocyte ghosts and rat brain synaptosomal membranes. A maleimide nitroxide probe (MAL-6) was coupled to membrane protein, or 5-doxylstearic (5NS) was dissolved in the membrane lipid, and the electron spin resonance (ESR) spectra were recorded in the presence of increasing ethanol concentrations and at different temperatures from 4 degrees to 37 degrees. The lipid probe at all temperatures studied showed an increase in fluidity in the membrane lipid when ethanol was added in concentration over 20 mM. The protein probe however required a high concentration of ethanol (200 mM) to produce an increase in the rigidity of the protein conformation of 4 degrees in both erythrocyte and synaptosomal membranes. A decrease in protein rigidity was observed at high ethanol concentrations at 37 degrees only in the erythrocytes. Thus an effect of ethanol on the conformation of membrane protein was observed using MAL-6 only at high ethanol concentrations and depended on the membrane used and the temperature of measurement.

Animals↗
PubMedDate not supplied

[Comparison of the heme electron state of reduced cytochrome P450 and P420 in equilibrium and non-equilibrium protein conformations. The nature of the protein heme ligand].

Magnetic circular dichroism (MCD) spectra of reduced cytochromes P450 and P420 in equilibrium and non-equilibrium protein conformations are compared at 4.2 K for the 350-800 spectral region. Non-equilibrium forms have been produced by photolysis of CO-complexes at 4.2 K. The differences between MCD spectra of proteins in equilibrium and non-equilibrium conformations, in particular for the visible region, show clearly the structural changes in the heme iron coordination sphere to occur on ligand binding. The comparison of the Soret MCD spectra of reduced proteins in their equilibrium and non-equilibrium forms with those of other high-spin ferrous hemoproteins suggest that mercaptide (RS-) is the protein ligand of the heme iron in reduced P450, as well as in its CO-complex, and that imidazole of histidine is the fifth ligand of the iron both in reduced P420 and its CO-complex. The thermal recombination of the photoproducts with CO have been studied. When temperature rises from 4.2 to 77 K for two hours both proteins have similar temperature characteristics during the recombination processes. The recombination begins at T approximately equal to 10 K and is completed at approximately equal to 50 K. The temperature at which half of the total photolyzed molecules are restored to the CO-form is equal to 25 K. For products of photolysis of CO-complexes of myoglobin and hemoglobin under the same heating conditions these temperatures are equal to 35 and 23 K respectively. Thus, the photoproducts of P450, P420 and hemoglobin have similar parameters of low-temperature recombination and the kinetics of this process is faster than for photodissociated myoglobin.

Animals↗
PubMed1995

Nucleotide hydrolysis and protein conformational changes in Azotobacter vinelandii nitrogenase iron protein: defining the function of aspartate 129.

The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 microM) was comparable to that found for wild-type Fe protein (128 microM). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 microM). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein (-290 mV for wild-type Fe protein and -300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wild-type and D129E Fe proteins shifted to -430 and -440 mV, respectively. The D129E Fe protein was also found to bind to the molybdenum-iron protein (MoFe protein) with normal affinity, although it could not support electron transfer to the MoFe protein or MoFe protein-stimulated MgATP hydrolysis.

2,2'-Dipyridyl↗
PubMed1973

Circular dichrosim of chromaffin granule proteins in situ: analysis of turbidity effects and protein conformation.

The circular dichroism spectra for proteins in situ in catecholamine secretory vesicles (chromaffin granules) is presented together with an analysis of protein conformation and turbidity effects on the spectra. The calculational analysis has resolved scattering and absorption effects in the turbid suspension spectra using a coated-sphere scattering model which allows for different materials in its shell and core. The intrinsic conformation of the proteins in situ was estimated by an iterative procedure with various trial protein conformations, for the chromaffin granules both intact and after release of their contents. The resulting average secondary structures (within about 10%) are: (25% alpha helix, 15% beta structure) for the membrane proteins and (15% alpha helix, 5% beta structure) for the soluble contents. The protein conformation did not change with osmotic release of the granule's contents. Consequently, if chromogranins are involved in a catecholamine storage complex, this is not reflected in any detectable change in their average secondary structure.

Cell Fractionation↗
PubMed2006

Fragment library-based representation system for protein conformation.

The representation system for protein conformation has a crucial effect on the speed of various protein-related simulations, including ab initio protein structure prediction and protein-protein docking simulation. Usually, the finer a representation system, the longer is the computational time required to employ the representation system in simulations. On the other hand, very coarse lattice systems cannot be directly applied to the simulation problems with real proteins. We report a new, fragment library-based protein conformation representation system, prepared by clustering amino acid conformations from 154 proteins. This system was composed of 64 most representative fragments per each amino acid, and based on the unified residue approach in which two spheres per amino acid were used. It could represent the conformation of the 82 proteins in an independent test set with the mean and standard deviation RMSD of 1.01 and 0.09 A, respectively, based on the position of alpha carbons and the centers of mass of sidechains.

Models, Molecular↗
PubMed1992

Dependence of reaction rate of 5,5'-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations.

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.

Dithionitrobenzoic Acid↗
PubMed1969

The influence of short-range interactions on protein conformation. 3. Dipeptide distributions in proteins of known sequence and structure.

A statistical analysis is made of the distribution into alpha-helical and non-alpha-helical regions of the various dipeptide types appearing in a sample of seven proteins of known sequence and structure. By considering as a group all dipeptide types occurring at a given location relative to the reported helix-coil boundaries and examining the percentage of cases in which these appear in non-alpha-helical regions throughout the protein sample, we find a sharp change in the nature of the observed dipeptide types when the helix-coil boundary is crossed. Furthermore, we find that dipeptide types which occur in the coil region near the C-terminal end of helical segments are non-alpha-helical in almost 90 per cent of the cases in which they appear throughout the sample. No similar effect is found in the coil region near the N-terminal end of helical segments. These results give evidence for the importance of short-range interactions in determining protein conformation. They are also consistent with predictions based on a model for helix formation given in the second paper of this series.(1)

Amino Acid Sequence↗
PubMed2005

Heat shock response modulators as therapeutic tools for diseases of protein conformation.

The disruption of protein folding quality control results in the accumulation of a non-native protein species that can form oligomers, aggregates, and inclusions indicative of neurodegenerative disease. Likewise for over 100 other human diseases of protein confirmation, a common feature may be the formation of off-pathway folding intermediates that are unstable, self-associate, and with time lead to a chronic imbalance in protein homeostasis with deleterious consequences on cellular function. This has led to a hypothesis that enhancement of components of the cellular quality control machinery, specifically the levels and activities of molecular chaperones, suppress aggregation and toxicity phenotypes to allow cellular function to be restored. This review addresses the regulation of molecular chaperones and components of protein homeostasis by heat shock transcription factor 1 (HSF1), the master stress-inducible regulator, and our current understanding of pharmacologically active small molecule regulators of the heat shock response as a therapeutic strategy for protein conformational diseases.

DNA-Binding Proteins↗