Perturbation Of Protein Structure Research Articles

Study of oat globulin conformation by Fourier transform infrared spectroscopy.

The conformation of oat globulin dispersions (10% in D2O) under the influence of pH, chaotropic salts, protein structure perturbants, and heating conditions was studied by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectrum of oat globulin showed major bands from 1670 to 1634 cm(-1), corresponding to the four major types of secondary structures, that is, beta-turns, beta-sheets, alpha-helices, and random coils. At extreme acidic and alkaline pH conditions, there were changes in intensity in the bands attributed to beta-sheet structures (1626, 1634, and 1682 cm(-1)), and shifts of the bands to higher or lower wavenumbers, indicating changes in conformation. In the presence of some chaotropic salts, the 1626 and 1634 cm(-1) bands were shifted upward, with a marked decrease in the intensity of the 1634 cm(-1) peak. The addition of several protein structure perturbants led to a slight shift in the alpha-helix/random coil bands and a marked reduction in the beta-sheet peaks, suggesting protein unfolding. Heating under aggregating conditions led to slight shifts in all of the major bands and progressive changes in the intensity of the alpha-helix, beta-sheet, and beta-turn peaks, suggesting protein denaturation. This was accompanied by marked increases in intensity of the two intermolecular beta-sheet bands (1682 and 1624-1626 cm(-1)) associated with the formation of aggregated strands. The IR spectra of soluble and insoluble aggregates showed a redistribution of native and extensively denatured proteins in the two fractions.

Prion protein interconversions.

The transmissible spongiform encephalopathies (TSEs), or prion diseases, remain mysterious neurodegenerative diseases that involve perturbations in prion protein (PrP) structure. This article summarizes our use of in vitro models to describe how PrP is converted to the disease-associated, protease-resistant form. These models reflect many important biological parameters of TSE diseases and have been used to identify inhibitors of the PrP conversion as lead compounds in the development of anti-TSE drugs.

Structural Changes of Cytochrome c552 from Thermus thermophilus Adsorbed on Anionic and Hydrophobic Surfaces Probed by FTIR and 2D-FTIR Spectroscopy

The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons.

Encapsulation of bovine serum albumin in poly(lactide-co-glycolide) microspheres by the solid-in-oil-in-water technique.

Non-aqueous protocols to encapsulate pharmaceutical proteins into biocompatible polymers have gained much attention because they allow for the minimization of procedure-induced protein structural perturbations. The aim of this study was to determine if these advantages could be extended to a semi-aqueous encapsulation procedure, namely the solid-in-oil-in-water (s/o/w) technique. The model protein bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by first suspending lyophilized BSA in methylene chloride containing PLG, followed by emulsification in a 1% aqueous solution of poly(vinyl alcohol). By variation of critical encapsulation parameters (homogenization intensity, BSA:PLG ratio, emulsifier concentration, ratio of organic to aqueous phase) an encapsulation efficiency of > 90% was achieved. The microspheres obtained showed an initial burst release of < 20%, a sustained release over a period of about 19 days, and a cumulative release of at least 90% of the encapsulated BSA. Different release profiles were observed when using different encapsulation protocols. These differences were related to differences in the microsphere erosion observed using scanning electron microscopy. Release of BSA was mainly due to simple diffusion or to both diffusion and microsphere erosion. Fourier-transform infrared studies were conducted to investigate the secondary structure of BSA during the encapsulation. Quantification of the alpha-helix and beta-sheet content as well as of overall structural changes showed that the secondary structure of encapsulated BSA was not more perturbed than in the lyophilized powder used initially. Thus, the encapsulation procedure did not cause detrimental structural perturbations in BSA. In summary, the results demonstrate that the s/o/w technique is an excellent alternative to the water-in-oil-in-water technique, which is still mainly used in the encapsulation of proteins in PLG microspheres.

Flow property of globulin from red bean ( Phaseolus angularis)

Red bean globulin (RBG) dispersions exhibited pseudoplastic behavior, and the flow data were fitted to the power-law model. With increasing protein concentration, there were progressive increases in consistency coefficient ( m), apparent viscosity and yield stress, suggesting protein–protein interactions. The flow behavior index ( n) was decreased indicating enhanced pseudoplasticity. Ionic strength affected the flow behavior of RBG, with a reversal in its effect at a specific salt concentration (0.5–1.0 M). Addition of chaotropic salts and protein structure perturbants (urea, sodium dodecyl sulfate and dithiothreitol) caused changes in flow behavior, probably due to dissociation of the oligomers and protein unfolding. Pre-heat treatments at a temperature of 80°C, above the onset temperature of RBG, caused dramatic decreases in n and increases in m and apparent viscosity suggesting protein denaturation/aggregation. When RBG dispersions were heated from 25 to 90°C at a fixed rate (1.7°C/min), there was an initial decrease in apparent viscosity, probably due to a lowering of medium viscosity. The viscosity started to increase at around 78°C, the onset temperature of RBG; and sharp increases were observed from 80 to 90°C. Changes in apparent viscosity with increasing temperature (25–60°C) obeyed the Arrhenius law, with similar E a values at three different shear rate values, suggesting little temperature dependence of viscosity at lower temperature range.

Protein folding and stability: What can we learn from high pressure?

The relationships between the amino acid sequence of proteins and their structural and functional specificity have yet to be elucidated, as do the determinants for the marginal stability of the folded form of the chain. The mechanisms by which proteins fold and the nature of the rate-determining step in folding remain important questions in the folding field. One important aspect of folded proteins that differentiates them from non-biological polymers is the compactness of their folded structures. Proteins, in fact, are packed as densely as crystals of small organic compounds. Thus the shape of each amino acid plays an important role in providing maximal contacts and interaction free energy within the specific folded structure. Deeper insight into the importance of packing efficiency in defining the form and stability of the folded structure can be gained by probing the volumetric properties of proteins. High pressure as a perturbation of protein structure can give access to such volumetric information concerning equilibrium or transition states in the folding process. The results of pressure denaturation studies must be interpreted in the context of other thermodynamic and kinetic data on protein folding in order to construct a global view of the folding process.

Perturbation of Lipid and Protein Structure by General Anesthetics: How Little Is Too Little?

Jonathan W. Tanner M.D., Ph.D.*Assistant Professor of AnesthesiaDepartment of AnesthesiaThe Johnson Research Foundationjohansso@mail.med.upenn.edu.Assistant Professor of Anesthesia Department of AnesthesiaUniversity of PennsylvaniaPhiladelphia, Pennsylvania 19104In Reply:—Dr. Raines correctly points out that the effects of either 1 MAC isoflurane or 1 MAC halothane on tryptophan side-chain mobility in bovine serum albumin is comparable to what follows from a 1°C reduction in temperature. Changes in lipid fluidity in the presence of anesthetic molecules can be mimicked by small variations in temperature, and this has been argued to indicate that lipids are an implausible site of anesthetic action. 1By analogy then, as noted by Dr. Raines, the same line of argument would suggest that a protein target would be an equally unlikely in vivo site of anesthetic action.If anesthetics interact directly with protein targets and alter their function, then binding must influence either the structure of the protein or its dynamics. Alternatively, anesthetics may compete with native ligands for their binding sites. 2The latter mode of action does not appear to apply in the case of ligand-gated ion channels, since anesthetics increase the affinity of the native neurotransmitter. 3The limited information on protein structural changes induced by bound anesthetic molecules indicates that secondary structure is not altered. 4–7It is therefore likely that a bound anesthetic molecule instead perturbs the tertiary structure of the protein, or perhaps the quaternary structure. Examples of the latter structural change are the effects of halothane and diethyl ether on the aggregation state of the membrane-bound Ca-ATPase. 8,9The 2.2 Å X-ray crystal structure of firefly luciferase with two bound bromoform molecules revealed that anesthetic binding caused minimal overall protein structural changes. 7Another possibility is that anesthetics may not alter protein structure but instead modify amino acid side-chain dynamics, 10which are intimately related to protein function. In line with changes in dynamics is the finding that bromoform binding caused a neighboring histidine residue (H310) in the firefly luciferase crystal structure to become less mobile. 7High resolution X-ray crystallography will be required to detect the small structural changes that are likely in the case of weakly interacting ligands such as the volatile general anesthetics. A promising alternative approach for examining the effect of a bound anesthetic molecule on protein structure and side-chain mobility is to perform molecular dynamics simulations. 11The effect of anesthetic agents on bovine serum albumin dynamics suggested a potential mechanism for how protein function is altered. It was proposed that anesthetic binding traps the protein in a substate with a lower free energy minimum than the native substate, and therefore effectively prevents the conformational changes required for normal protein activity at a physiologic temperature. 10Studies with additional proteins will reveal whether this model has wide applicability. It certainly remains plausible that physical properties of lipids and proteins not amenable to experimental analysis at present are responsible for the clinical effects of inhaled anesthetics. One example of this is the lipid-based mechanism of general anesthetic action proposed by Cantor. 12However, until such theories can be experimentally tested, we remain optimistic that currently available biophysical tools will provide useful information, and generate testable hypotheses, regarding how these important clinical agents may exert their effects on the central nervous system.

Perturbation of Lipid and Protein Structure by General Anesthetics: How Little Is Too Little?

Assistant Professor of Anesthesia Harvard Medical SchoolAssistant AnesthetistMassachusetts General HospitalDepartment of AnesthesiaBoston, Massachusetts 02114Raines@etherdome.MGH.harvard.edu.To the Editor:—Lipid-based theories of general anesthetic action have long endured because numerous studies have shown that the in vivo pharmacology of an anesthetic correlates remarkably well with its ability to perturb the structural properties of simple lipid bilayers. The Meyer-Overton correlation between anesthetic potency and hydrophobicity, the inactivity of nonanesthetic long chain alcohols and highly halogenated volatile compounds (nonimmobilizers), and pressure reversal have all been demonstrated in studies using protein-free lipid bilayers. 1–6Nevertheless, a most persuasive and often mentioned argument against lipid-based theories is that at clinically relevant concentrations, anesthetics induce only small perturbations in lipid bilayer structure. 7,8For example, halothane reduces the order parameter (increase the “fluidity”) of lipid bilayers by only 1% at clinically relevant concentrations. 9An equivalent reduction in order parameter may be obtained by raising the temperature of the bilayer by less than 1°C. Similarly, halothane reduces the transition temperature between a lipid bilayer’s liquid and gel phases by only 0.5°C at anesthetic concentrations and by only 5°C even at 10 times the minimum aveolar concentration (MAC). 10I was, therefore, very interested to read the study by Johansson et al. quantifying the effects of isoflurane and halothane on structural properties of bovine serum albumin, a lipid-free protein model used in mechanistic studies of anesthetic action. 11What did their studies show? At approximately 1 MAC, isoflurane and halothane increased the fluorescence anisotropy of bovine serum albumin by 1%. An equivalent reduction was obtained by raising the temperature of bovine serum albumin by approximately 1° C. Similarly, isoflurane and halothane raised the transition temperature between the folded and unfolded states of bovine serum albumin by less than 1°C at anesthetic concentrations and by only 3–4°C even at 10 times MAC. Studies of anesthetic binding to other protein models have been similarly unable to demonstrate significant anesthetic-induced changes in protein structure. 12,13Thus, anesthetics induce similarly small changes in the structural properties of lipids and proteins. For consistency, shouldn’t we now conclude that such insensitivity argues strongly against a protein site of anesthetic action?The inability to detect significant anesthetic-induced structural changes in either lipid or protein model systems highlights the practical (and obvious) limitations of such studies: we can only measure what we can measure. Fluorescence anisotropy, denaturation temperature, phase transition temperature, and order parameter have been used by biophysicists for many years as indicators of lipid bilayer and protein structure in large part because they are easily quantitated. There is no compelling theoretical reason to believe that changes in these properties directly accounts for the functional effects of anesthetics on relevant targets in the central nervous system. In fact, it seems quite likely that the anesthetic state results from changes in other lipid and/or protein physical properties that are not so easily measured.

Identification of Two Major Sites in the Type I Interleukin-1 Receptor Cytoplasmic Region Responsible for Coupling to Pro-inflammatory Signaling Pathways

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: dehydration stress in the presence of sucrose.

First, to investigate the role of sucrose in stabilizing protein structure (as measured by changes in the amide I band of lysozyme) caused by dehydration encountered during lyophilization. Second, to demonstrate the utility of internal reflection spectroscopy as a tool for conducting controlled lyophilization experiments. A custom-built internal reflection FTIR accessory was used to follow the entire freeze-drying process of solutions consisting of 49.4 mg/mL lysozyme in the presence and absence of 10% sucrose in real-time. Studies were carried out using D2O as a transparent medium in the infrared region of the protein amide bands. Potential self-association of the protein in the presence of sucrose was investigated using dynamic light scattering. Hydration levels were determined using a multiple regression equation. Differential scanning calorimetry (DSC) permitted characterization of the final lyophilized product. Moisture content was determined using Karl Fischer titration. Throughout freezing and drying, minimal changes were observed both in frequency (1647 +/- 1 cm-1) and bandwidth (46 +/- 1 cm-1) of the amide I band in the presence of sucrose. In contrast, greater changes in frequency and band width were seen in the absence of sucrose. A successfully lyophilized cake was obtained which had properties of a glass as measured by DSC, with a Tg of 50 degree C. The lyophilized product containing sucrose had 4% moisture by weight. Three distinct rates of water desorption were discovered during drying under vacuum (50 mg/hr within the sample temperature range from -35 degrees to -25 degrees C; 30 mg/hr from -10 degrees to 25 degrees C; 1.2 mg/hr from 27 degrees to 38 degrees C). The inclusion of sucrose served to minimize perturbations of protein structure caused by freezing and dehydration stresses encountered during lyophilization (compared to studies conducted in absence of sucrose). The results support the water replacement hypothesis and underscore the role of the sugar in preserving a native structure in the dried state. This investigation demonstrates the usefulness of infrared spectroscopy in evaluating lyophilization process parameters and formulation design.

Secondary Structure and Temperature-induced Unfolding and Refolding of Ribonuclease T1 in Aqueous Solution: A Fourier Transform Infrared Spectroscopic Study

Max-Delbrück-Centrum für Molekulare Medizin Robert-Rössle-Straße 10, D-1115 Berlin-Buch, Germany; Robert-Koch Institut des Bundesgesundheitsamtes Nordufer 20, D-1000 Berlin 65, Germany and Institut für Kristallographie der Freien Universität Berlin Takustraße 6, D-1000 Berlin 33, GermanyThe secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% α-helix and 32 to 33% β-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with the crystal structure which indicates 16% α-helix and 35% β-sheet. Specific IR-marker bands are assigned to the different β-sheet structures, to the slightly bent α-helix, and to β-turn and irregular conformations present in RNase T1. The temperature dependence of the infrared spectra shows that the thermal unfolding and refolding of RNase T1 is fully reversible. This permitted the detailed analysis of structural changes that occur as a function of temperature by evaluating quantitatively the various secondary structure-related amide I band components and some amino acid side-chain vibrations as specific monitors. The secondary structure of RNase T1 is essentially retained in the temperature range between 20 and 50°C. Significant perturbation of protein structure is initiated between 50 and 55°C within regions of β-sheet structures while the α-helix remains virtually intact up to 55°C suggesting a "premelting" of RNase T1. Between 55 and 60°C, a highly co-operative unfolding process is indicated by the simultaneous breakdown of all secondary structure components and by distinct changes of some specific side-chain vibrations. An analysis of the amide I band contour of RNase T1 at 70°C proves that the unfolded state is predominantly, but not completely, irregular or "random coil". Residual, turn-like structures persisting even in the unfolded state are suggested by minor, turn related band components in the amide I region. From IR-spectra collected along a linear temperature gradient, intensity/temperature and frequency/temperature profiles were constructed using some peptide backbone and amino acid side-chain marker bands as local, structure-sensitive monitors. From these profiles individual transition temperatures tm and transition enthalpies ΔH (van't Hoff) were calculated. The tm and ΔH values revealed a small but distinct hysteresis between repetitive cycles of unfolding and refolding of the protein, suggesting slow refolding kinetics of RNase T1. Furthermore, the various infrared "marker bands" indicate a slightly different response towards temperature increase/decrease for different regions of the protein. The data demonstrate that infrared spectroscopy permits both the detailed analysis of structural changes occurring in a protein as a function of temperature and the determination of thermodynamic parameters characterizing its folded/unfolded state transition.

Phosphorescence spectra of R-phycoerythrin

The phosphorescence of R-phycoerythrin from the red alga Callithamnion corymbosum was detected. The phosphorescence spectrum of R-phycoerythrin has two main components with maxima at 720 nm and 820 nm and lifetimes of 2.5 ms and 6.0 ms respectively. The phosphorescence excitation spectrum exhibited three bands at 498, 541 and 566 nm, characteristic of the absorption spectrum of R-phycoerythrin, but their relative intensifies differed from those recorded in fluorescence excitation spectrum. The fluorescence quantum yield of the pigment was 0.8 at 77 K; the phosphorescence quantum yield was 1.6 × 10 −6 and it increased to 1.5 × 10 −4 for the R-phycoerythrin denatured with 8 M urea at pH 3.0. This might be explained partly by alteration of the chromophore conformation accompanying the perturbation of protein structure.

Asymmetric fusion between synthetic di-n-dodecylphosphate vesicles and virus membranes

The interaction between vesicles, prepared from the synthetic amphiphile di-n-dodecylphosphate (DDP), with Sendai virus membranes was investigated. DDP vesicles fuse in the presence of Ca 2+ (‘symmetric’ fusion). However, in the absence of Ca 2+, DDP vesicles and Sendai virus, both displaying a high intrinsic fusion capacity with various target membranes, can also readily fuse with each other (‘asymmetric’ fusion). Under these conditions, fusion was found not to depend on specific viral proteins. Thus fusion occurs over a broad pH range (3.0–9.0) and is not affected by perturbation of viral protein structure. The overall interaction process was further analyzed with a mass action kinetic model. The analysis reveals that the destabilization and reorganization of the synthetic and viral bilayers are as fast as in pure phospholipid systems. Furthermore, the drastic effect of temperature on the overall reaction appears to be related to an effect of this parameter on fusion itself rather than on vesicle-virus aggregation. This could suggest that protein mobility constraints modulate the fusion reaction. The morphology of the fusion products, which consist of a single virus particle and several DDP vesicles, indicates a bilayer stabilization of the fusion product, rather than formation of tubular structures, as observed for symmetric DDP fusion products. The present results further emphasize the high susceptibility of vesicles composed of synthetic amphiphiles to engage in (protein-mediated) membrane fusion. This bears relevance to their potential application as carriers for biomolecules.

Modeling chemoadaptive behavior: X-ray structure of the trimethylamine oxide-urea (1?4) complex

The structure of a trimethylamine oxide (TMAO)-urea 1∶4 complex was investigated by x-ray crystallographic methods. C3H9NO, 4CH4N2O, Mr=315.33, monoclinic P21/n, a=11.004(2), b=9.8259, c=15.419(2),Β=106.88(4)‡, V=1595.4(2) a3, Z=4, Dx=1.313(1) g cm−3, monochromatized Cu λα (λ=1.5418 a),Μ=8.94 cm−1, F(OOO)=680, temperature = 110‡K, final R=0.067, for 1786 unique observed reflections. Based on these studies and comparison with related compounds a model of the interaction of urea and TMAO with proteins, especially enzymes, is proposed. This is important in the physiology of many organisms, especially elasmobranchs. The counteracting effect of TMAO on urea perturbation of protein structure and function is interpreted in terms of water structure as a balance between the structure breaking effect of urea and the structure-stabilizing effect of TMAO.

Possible process of insolubilization of lens proteins—Direct effect of glucose

The effect of glucose on the insolubilization of lens proteins was investigated in the presence of visible light. The amount of glucose-induced insolubilization of lens proteins showed a dose-dependent increase. The velocity of insolubilization accelerated under acidic conditions. These results suggest that this phenomenon may be due to the direct action of glucose rather than glycosylation. 1H-NMR spectroscopy results suggest that the action of glucose might be to induce conformational changes, and aggregation of the proteins. The in vitro observation of glucose-derived insolubilization of the lens proteins may be similar to that observed in diabetic mellitus. Elevated free glucose per se may have a role in diabetic cataractogenesis in addition to the metabolism of glucose to sorbitol and osmotic changes to the lens. Protein structural perturbation by glucose was demonstrated with isolated proteins from the aged normal human lenses over 40 years and may contribute to senile cataractogenesis. The results suggest that elevated lens glucose may act directly as one of the triggers of cataractogenesis.

Solubility-activity relationships in the inorganic salt-induced precipitation of α-chymotrypsin

Inorganic salt-induced precipitation is a commonly employed protein separation/concentration technique that is potentially denaturing. The effects of precipitating environments on recoverable activity were investigated using α-chymotrypsin as a model protein. The following salts, in order of increasing denaturation potential and decreasing molal surface tension increment, were studied: Na 2SO 4, NaCl, NaBr, KBr, and KSCN. These salts span the lyotropic series. Solubility measurements gave salting-out constants that disagree with current salting-out theory. Salt-protein interactions and concomitant protein structural perturbations are the likely source of the discrepancies. Activity and active fraction measurements indicated that the fraction of precipitate that is active upon dissolution is a function of the salt molal surface tension increment, but not the salt concentration. The specific activity of soluble precipitate remained essentially constant regardless of salt concentration or type. A salting-out performance parameter was defined and calculations indicated that an optimum salt may exist for a particular protein. The recovered active fraction was not affected by varying the amount of salt added and, correspondingly, the amount of protein precipitated.

Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca 2+, or with P i, in the absence of Ca 2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of P i binding and enzyme phosphorylation with P i (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the P i and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of p i and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.

Denaturation of a membrane transport protein by urea: the erythrocyte anion exchanger.

Chloride equilibrium exchange was measured in the presence of intracellular and extracellular urea, several different alkylureas and thiourea. Urea half-inhibited Cl exchange at about 2.5 M, but the other, less polar analogs had significantly higher potencies; e.g., butylurea half-inhibited at about 60 mM. Onset and reversal of inhibition occurred within less than 2 sec. The inhibition exhibited no obvious sigmoidal dependence on urea concentration, and at low concentrations dimethylurea was a noncompetitive inhibitor of Cl exchange. However, at higher concentrations the Dixon plots were curved upward and a Hill analysis of the dimethylurea data yielded a Hill coefficient of at least 1.5. When present on only one side of the membrane, the slowly penetrating thiourea inhibited Cl exchange with a higher potency from the outside of the cell. Cl/Br exchange was inhibited less under conditions of self-inhibition of anion exchange than in the absence of self-inhibition. These data indicate that the ureas inactivate the anion transporter by a reversible denaturation process, and that the function of the anion transport mechanism may be more sensitive to small perturbations of protein structure than are spectroscopically derived structural parameters.

Reactivation of glutamine synthetase from Escherichia coli after auto-inactivation with L-methionine-S-sulfoximine, ATP, and Mn2+.

Escherichia coli glutamine synthetase auto-inactivated with L-methionine-S-sulfoximine and ATP can be completely reactivated at pH 3.5-4.6 in 1 M KCl and 0.4 M (NH4)2SO4. Both unadenylylated and adenylylated magnesium and manganese enzymes can be reactivated. Reactivation of fully inactivated enzyme is first order (t1/2 approximately 2 min at pH 4.1 and 37 degrees C) and coincides with the stoichiometric release of 0.95 +/- 0.05 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2.0 +/- 0.2 eq of Mn2+ from each subunit. The rate of reactivation increases with decreasing pH and is proportional to the 3rd to 4th power of the hydrogen ion activity; the protonation of 3-4 carboxylic acid groups/subunit therefore may be required to disrupt the enzyme complex. Reactivation rate also increases with increasing KCl concentrations and temperature, with an Arrhenius activation energy of approximately 26 Kcal/mol, suggesting that some protein structural perturbation is required to disrupt the complex. Upon neutralization os reactivation solutions, the ligands and metal ions recombine with the enzyme resulting in its complete reinactivation. Thus, inactivation of glutamine synthetase at neutral pH is due to the extremely tight, but reversible, binding of L-methionine-S-sulfoximine phosphate, ADP, and Mn2+ (K'A greater than 10(12) M-1 for ADP) to the enzyme. Under certain conditions, ATP or ADP can partially inhibit inactivation. Excess ADP inhibits reinactivation by L-methionine-S-sulfoximine phosphate, and ATP has a complex effect on the rate and extent of the autoinactivation reaction.

Some regularities of dynamic accessibility of buried fluorescent residues to external quenchers in proteins

The temperature and viscosity dependences of quenching of buried tryptophan residues of several proteins by external ionic (iodide) and neutral (acrylamide) quenchers have been studied. The effective quenching rate constant is shown to be proportional to the diffusion coefficient of the free solvent (water or 20 vol% glycerol). This fact supports the idea that the accessibility of buried chromophores of proteins to quenchers is based on the dynamic perturbations in protein structure (the dynamic accessibility). These structural perturbations are assumed to be governed by some diffusion-limited processes in the solvent surrounding the protein molecule.