Crystal structure of hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres

The crystal structure of tetragonal hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres has been determined by X-ray diffraction to a nominal resolution of 2 A. The crystals, originally grown in 0.83 M-NaCl, had to be transferred to 1.4 M-NaCl to prevent crystal cracking at 300 to 400 atm. The a and b axes of the unit cell contracted by 0.6%, whilst the c axis increased by 0.1%. The unit cell volume contracted by 1.1%. Both the 1 atm and the 1000 atm structures were refined by restrained least-squares to yield final R factors of 14.9% in each case. Since the data were collected by an accurate difference protocol, the change in structure is considered to be more accurate than the absolute structure. The probable accuracy of the atomic shifts is shown to be +/- 0.06 A. The estimated volume decrease of the whole molecule corresponded to an isothermal compressibility of 4.7 X 10(-3) kbar-1. The contraction was non-uniformly distributed. Domain 2 (residues 40 to 88) was essentially incompressible, whilst domain 1 (residues 1 to 39, 89 to 129) had a compressibility of 5.7 X 10(-3) kbar-1. The interdomain region was also compressible. The average B factor decreased about 1 A2 at 1000 atm, but there was a wide range of decreases and increases in individual values. The pressure-induced deformation was analyzed with difference distance matrices. The beta-sheet (residues 42 to 60) and helix 2 (residues 24 to 36) were deformed the least under pressure. The other helices were more deformed and one loop region (residues 61 to 87) actually appeared to expand. The main-chain atoms of the beta-sheet and helix 2 were used to perform a least-squares superposition of the 1 atm and 1000 atm models. The root-mean-square pressure-induced shift for all atoms was 0.2 A, with a few atoms moving more than 1 A. There was no evidence for co-ordinated movement about the hinge axis defined by alpha carbon atoms 38 and 97. The 1 atm and 1000 atm refined structures included 151 and 163 ordered water molecules, respectively. The changes in these ordered water molecules and the mean compressibility of all of the solvent in the crystal will be described elsewhere.     

Effect of hydrostatic pressure on the solvent in crystals of hen egg-white lysozyme

The mass density of protein crystals can be measured in Ficoll gradients as a function of hydrostatic pressure. Carbon tetrachloride-toluene mixtures provide convenient density markers, and the compressibility of these standards is reported. Measurements on tetragonal crystals of hen egg-white lysozyme yielded densities at room temperature of 1.2367(+/- 0.0010) g cm-3 at 1 atm and 1.2586(+/- 0.0017) g cm-3 at 1000 atm (1 atm = 101,325 Pa). When combined with the unit cell dimensions at these two pressures these values lead to an estimated compression (fractional change in volume) of the crystal solvent at 1000 atm of 0.0369(+/- 0.0054). This value is comparable to that of a 0.7 M solution of NaCl. From an approximate estimate of the Donnan effect for the crystal in the 1.4 M-NaCl mother liquor, the crystal solvent contains 0.8 M-Na+ and 2.5 M-Cl-. It is concluded that the compressibility of solvent in lysozyme crystals is, within experimental error, the same as bulk solvent and does not exhibit the dramatically altered compressibility expected of an ice or glass-like solid. The crystallographically observable water sites, 151 at 1 atm and 163 at 1000 atm, showed a tendency to increase the number of hydrogen bonds made to other water sites at the expense of hydrogen bonds made to protein. The explanation for this phenomenon is presently unknown. Water sites that occur in both structures tend to have comparable temperature factors and show some tendency to follow the pressure-induced changes in protein atom positions. The compression expected for the water molecules themselves is too small to be observable at the resolution of the X-ray data collected in this study.     

Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

Pressure effects on actin self-assembly: interspecific differences in the equilibrium and kinetics of the G to F transformation

Purified skeletal muscle actins from species whose ambient pressures range from 1 to greater than 500 atm were examined for the sensitivity to hydrostatic pressure of the globular (G) to filamentous (F) self-assembly reaction. Both the equilibrium position and the kinetics of self-assembly were affected by pressure. Increased pressure shifted the self-assembly equilibrium toward the monomer (G) state and reduced the rate of F-actin assembly. For most of the actins studied, the perturbation by pressure of F-actin formation decreased with increasing measurement of pressure, indicating that F-actin has a higher compressibility than G-actin. The increase in system volume and compressibility concomitant with the assembly of F-actin can be interpreted as reflections of the major role played by hydrophobic effects in stabilizing F-actin and of the existence of "hard" binding sites, in the terminology of Torgerson et al. [Torgerson, P. M., Drickamer, H. G., & Weber, G. (1979) Biochemistry 18, 3079-3083], in the actin subunits. For actin from the deepest occurring species studied, the teleost fish Coryphaenoides armatus, which occurs to depths of approximately 5000 m (equivalent to 501 atm of pressure), there was no difference in compressibility between G-actin and F-actin; that is, the effect of increasing pressure on self-assembly was linear over the entire pressure range examined, 600 atm. The self-assembly reaction of the actin from C. armatus also differed from that of the other actins examined in that the G to F equilibrium was relatively insensitive to increased pressure; i.e., the volume change (delta V) of assembly was small.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure-adaptive differences in proteolytic inactivation of M4-lactate dehydrogenase homologues from marine fishes

The inactivation by hydrostatic pressure of muscle-type lactate dehydrogenase (M4-LDH, EC 1.1.1.27; L-lactate: NAD+ oxidoreductase) homologues from five shallow-living and six deep-living marine teleost fishes was compared. The pressures which inactivate these enzymes are much higher than the pressures experienced by any of the species. To determine whether hydrostatic pressure effects on protein aggregation state and conformation might influence proteolysis, the inactivation of LDH by the proteases, trypsin (EC 3.4.21.4) and subtilisin (EC 3.4.4.16) was determined at atmospheric pressure and 1,000 atm pressure. At 10 degrees C and atmospheric pressure, the enzymes of the shallow-living fishes are inactivated four times faster by trypsin and three times faster by subtilisin than are the homologues of the deep-living species. At 1,000 atm pressure, the homologues of shallow-occurring fishes were inactivated 28 to 64% more than predicted from the summed effects of denaturation by 1,000 atm pressure and tryptic inactivation at atmospheric pressure. In contrast, the homologues of the deep-sea species were inactivated by trypsin 0 to 21% more than expected. At 1,000 atm, inactivation by subtilisin increased to a similar degree for enzymes from both deep- and shallow-living species. However, at 1,000 atm, the M4-LDH homologues of the deep-sea species lost less activity (55.3%) than did the homologues of the shallow species (86.4%). In comparisons made at 200 atm, a pressure typical of the habitat of the deep-occurring species, tryptic inactivation of the LDH of the shallow-living Sebastes melanops was increased 14%. No pressure inactivation of the enzyme is evident at 200 atm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Harmonicity and anharmonicity in protein dynamics: A normal mode analysis and principal component analysis

A comparison of a normal mode analysis and principal component analysis of a 200-ps molecular dynamics trajectory of bovine pancreatic trypsin inhibitor in vacuum has been made in order to further elucidate the harmonic and anharmonic aspects in the dynamics of proteins. An anharmonicity factor is defined which measures the degree of anharmonicity in the modes, be they principal modes or normal modes, and it is shown that the principal mode system naturally divides into anharmonic modes with peak frequencies below 80 cm^?1, and harmonic modes with frequencies above this value. In general the larger the mean-square fluctuation of a principal mode, the greater the degree of anharmonicity in its motion. The anharmonic modes represent only 12% of the total number of variables, but account for 98% of the total mean-square fluctuation. The transitional nature of the anharmonic motion is demonstrated. The results strongly suggest that in a large subspace, the free energy surface, as probed by the simulation, is approximated by a multi-dimensional parabola which is just a resealed version of the parabola corresponding to the harmonic approximation to the conformational energy surface at a single minimum. After 200 ps, the resealing factor, termed the “normal mode resealing factor,” has apparently converged to a value whereby the mean-square fluctuation within the subspace is about twice that predicted by the normal mode analysis. © 1995 Wiley-Liss, Inc.     

15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.     

Pressure-Dependent Structure Changes in Barnase on Ligand Binding Reveal Intermediate Rate Fluctuations

In this work we measured ¹H NMR chemical shifts for the ribonuclease barnase at pressures from 3 MPa to 200 MPa, both free and bound to d(CGAC). Shift changes with pressure were used as restraints to determine the change in structure with pressure. Free barnase is compressed by ∼0.7%. The largest changes are on the ligand-binding face close to Lys-27, which is the recognition site for the cleaved phosphate bond. This part of the protein also contains the buried water molecules. In the presence of d(CGAC), the compressibility is reduced by ∼70% and the region of structural change is altered: the ligand-binding face is now almost incompressible, whereas changes occur at the opposite face. Because compressibility is proportional to mean square volume fluctuation, we conclude that in free barnase, volume fluctuation is largest close to the active site, but when the inhibitor is bound, the fluctuations become much smaller and are located mainly on the opposite face. The timescale of the fluctuations is nanoseconds to microseconds, consistent with the degree of ordering required for the fluctuations, which are intermediate between rapid uncorrelated side-chain dynamics and slow conformational transitions. The high-pressure technique is therefore useful for characterizing motions on this relatively inaccessible timescale.     

Dynamics of Hemoglobin Investigated by Mössbauer Spectroscopy

Abstract

Mössbauer Spectra of ⁵⁷Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-Mössbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-Mössbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solutions above 200 K where the Lamb-Mössbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.     

Protein dynamics in an intermediate state of myoglobin: optical absorption, resonance Raman spectroscopy, and x-ray structure analysis

A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K or by electron transfer from photoexcited tris(2, 2'-bipyridine)-ruthenium(II) at 20 K. At temperatures above 150 K, the conformational transition toward the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy show that the metastable state has a six-ligated iron low-spin center. The x-ray structure at 115K proves the similarity of the metastable state with metmyoglobin. The Raman spectra yield the high-frequency vibronic modes and give additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at approximately 120 K. Parameters representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynamics are close to deoxymyoglobin. Thus within the metastable state the relaxation to the equilibrium is initiated by changes in the dynamic properties of the active site.     

Molecular dynamics simulation of solvated protein at high pressure.

We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure effects on sarcoplasmic reticulum

The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800-2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+-ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure.     

Molecular dynamics simulation shows large volume fluctuations of proteins

In this paper we present a new approach to study the volume fluctuations of proteins. From a 1 ns molecular dynamics simulation, the volume fluctuation of human lysozyme has been calculated. We used two different ways for the calculation. In the first one, the volume fluctuation is extracted directly from the trajectory. For the second one, a newly developed formalism based on principal component analysis is used. The r.m.s. volume fluctuations obtained from the two analyses agree well with each other. The isothermal intrinsic compressibility was found to be larger than the one reported by experiment. The difference is discussed and suggested to exist in the assumed uncertainty of the compressibility of hydrated water to deduce the isothermal intrinsic compressibility from the experimental value. Spectral analysis shows that low-frequency dynamics dominate the total volume fluctuation. The same aspect is found in the study using principal component analysis. This low-frequency region is related to large and slow motions of proteins. Therefore a long time dynamics simulation is necessary to describe the volume fluctuations of proteins.     

Comparison of normal mode analyses on a small globular protein in dihedral angle space and Cartesian coordinate space

Normal mode analyses on the protein, bovine pancreatic trypsin inhibitor, in dihedral angle space and Cartesian coordinate space are compared. In Cartesian coordinate space it is found that modes of frequencies lower than 30 cm(-1) contribute 80% of the total mean-square fluctuation and are represented almost completely by motions in the dihedral angles. Bond angle and length fluctuations dominate in modes above 200 cm(-1), but contribute less than 2% to the total mean-square fluctuation. In the low-frequency modes a good correspondence between patterns of atomic displacements was found, but on average the root-mean-square fluctuations of the Cartesian coordinate modes are 13% greater than their dihedral angle counterparts. The main effect of fluctuations in the bond angles and lengths, therefore, is to allow the dihedral angles to become more flexible. As the important subspaces determined from the two methods overlap considerably, dihedral angle space analysis can be applied to proteins too large for Cartesian coordinate space analysis.     

Characterization of dual-wavelength seminaphthofluorescein and seminapthorhodafluor dyes for pH sensing under high hydrostatic pressures

Hydrostatic pressure is an important physical parameter in biology, with pressures in the few-hundred-atm range having significant effects on cellular morphology, metabolism, and viability. To ensure valid results when studying pressure effects using fluorescence spectroscopy and imaging methods, metabolic probes need to be characterized for high-pressure use. Of interest is the sensing of pH at high pressures due to the key role that pH plays in cellular function. Despite the availability of pH-sensitive dyes, only a few have been characterized for high-pressure use. Here we present the effects of pressure on the acid-base equilibria of four dual-wavelength seminaphthorhodafluor and seminaphthofluorescein dyes (pK(a)=6.6-7.8). Using phosphate buffers as high-pressure pH references, we investigate the pressure dependence of pK(a) for these dyes and determine the volume change associated with the acid-dissociation reaction. We find that if pressure-induced pK(a) changes are not accounted for during interpretation of emission spectra, systematic errors of up to 0.02 pH units per 100atm would result, comparable to previously measured pressure-induced pH changes in vivo. Results are validated by correctly sensing pH changes in Tris and acetate solutions. Methods presented here are applicable to other metabolic probes utilizing dual-wavelength ratiometric sensing modes.     

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.     

Dissociation of peripheral protein-membrane complexes by high pressure.

The ability of high pressure to dissociate several peripheral protein-membrane complexes was investigated. Three vitamin K-dependent proteins (factor X, protein Z, and prothrombin) dissociated from small unilamellar vesicles (SUVs, 30 nm diameter) composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC) at comparable pressures (midpoints of 0.3-0.6 kbar). The pressure-induced dissociation curves for the factor X-SUV interaction followed the expected behavior for an interaction with an apparent dissociation equilibrium constant at atmospheric pressure, KD(atm), of 9 x 10(-7) M and a change in volume of association, delta Va, of 88 mL/mol. Factor X also dissociated from large unilamellar vesicles (LUVs, 100 nm diameter, 25% PS:75% PC) with a midpoint of 0.5 kbar. A second group of calcium-dependent membrane-binding proteins included protein kinase C (PKC), a 64-kDa protein, and a 32-kDa protein. The 32-kDa protein dissociated from SUVs (midpoint of 0.8 kbar), whereas PKC and the 64-kDa protein did not dissociate to a significant degree. The differences in dissociability of these proteins appeared to be a result of the differences in their KD(atm)'s (decreased dissociability with decreased KD(atm)). This pattern was further demonstrated by the relatively high midpoint of dissociation (1.1-1.4 kbar) of serum amyloid P component (SAP; KD(atm) ca. 10(-11)) and the limited dissociation of factor Va light chain (KD(atm) ca. 10(-11)). Changing the vesicle composition to phosphatidylethanolamine in place of PC gave higher affinity and decreased dissociation of the 32-kDa protein and SAP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear and non-linear pressure dependence of enzyme catalytic parameters

The pressure dependence of enzyme catalytic parameters allows volume changes associated with substrate binding and activation volumes for the chemical steps to be determined. Because catalytic constants are composite parameters, elementary volume change contributions can be calculated from the pressure differentiation of kinetic constants. Linear and non-linear pressure-dependence of single-step enzyme reactions and steady-state catalytic parameters can be observed. Non-linearity can be interpreted either in terms of interdependence between the pressure and other environmental parameters (i.e., temperature, solvent composition, pH), pressure-induced enzyme unfolding, compressibility changes and pressure-induced rate limiting changes. These different situations are illustrated with several examples.     

Cavity as a Source of Conformational Fluctuation and High-Energy State: High-Pressure NMR Study of a Cavity-Enlarged Mutant of T4Lysozyme

Although the structure, function, conformational dynamics, and controlled thermodynamics of proteins are manifested by their corresponding amino acid sequences, the natural rules for molecular design and their corresponding interplay remain obscure. In this study, we focused on the role of internal cavities of proteins in conformational dynamics. We investigated the pressure-induced responses from the cavity-enlarged L99A mutant of T4 lysozyme, using high-pressure NMR spectroscopy. The signal intensities of the methyl groups in the ¹H/¹³C heteronuclear single quantum correlation spectra, particularly those around the enlarged cavity, decreased with the increasing pressure, and disappeared at 200 MPa, without the appearance of new resonances, thus indicating the presence of heterogeneous conformations around the cavity within the ground state ensemble. Above 200 MPa, the signal intensities of >20 methyl groups gradually decreased with the increasing pressure, without the appearance of new resonances. Interestingly, these residues closely matched those sensing a large conformational change between the ground- and high-energy states, at atmospheric pressure. ¹³C and ¹H NMR line-shape simulations showed that the pressure-induced loss in the peak intensity could be explained by the increase in the high-energy state population. In this high-energy state, the aromatic side chain of F114 gets flipped into the enlarged cavity. The accommodation of the phenylalanine ring into the efficiently packed cavity may decrease the partial molar volume of the high-energy state, relative to the ground state. We suggest that the enlarged cavity is involved in the conformational transition to high-energy states and in the volume fluctuation of the ground state.