Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 degrees C and at various pressure values. The activation energy values were 181+/-8 kJ mol(-1) and 188+/-6 kJ mol(-1) for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 degrees C giving values around 100-125 ml mol(-1) which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.     

Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 °C and at various pressure values. The activation energy values were 181 ± 8 kJ mol^− 1 and 188 ± 6 kJ mol^− 1 for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 °C giving values around 100-125 ml mol^− 1 which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.     

Anion exchange in human erythrocytes has a large activation volume

Sulfate equilibrium exchange in human red cells has an activation volume of +150 +/- 20 cm3/mol over the pressure range 0.1 to 83 MPa (15 to 12000 lb/in2) at 30 degrees C. This value greatly exceeds the expected contribution from sulfate binding to the anion exchanger. We suggest that the activation volume reflects conformational changes during the transport cycle.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.     

Characterization of cold- and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens strain N6

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.     

Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids

The molecular organization of cholesterol in 1,2-didocosahexaenoylphosphatidylcholine (22:6-22:6PC) and 1-stearoyl-2-docosahexaenoylphosphatidylcholine (18:0-22:6PC) bilayers was investigated. Using low- and wide-angle X-ray diffraction (XRD), we determined that the solubility of the sterol at 20 degrees C was 11 +/- 3 mol % in 22:6-22:6PC vs 55 +/- 3 mol % in 18:0-22:6PC bilayers. Solubility in the dipolyunsaturated membrane rose to 17 +/- 3 mol % at 40 degrees C, while in the saturated-polyunsaturated membrane there was no change within experimental uncertainty. We compared the molecular orientation of [3alpha-(2)H(1)]cholesterol incorporated into 22:6-22:6PC bilayers to its solubility limit and into 18:0-22:6PC bilayers to a comparable concentration (10 mol %) in solid-state (2)H NMR experiments. The sterol possessed a tilt angle alpha(0) = 24 degrees +/- 1 degrees in 22:6-22:6PC that was independent of temperature over a range from 20 to 40 degrees C. In contrast, the value was alpha(0) = 21 degrees +/- 1 degrees in 18:0-22:6 bilayers at 20 degrees C and increased to alpha(0) = 24 degrees +/- 1 degrees at 40 degrees C. We attribute the low solubility of cholesterol in 22:6-22:6PC membranes to steric incompatibility between the rigid steroid moiety and the highly disordered docosahexaenoic acid (DHA) chain, which has the potential to promote lateral heterogeneity within DHA-rich membranes. Considering 22:6-22:6PC to be the most unsaturated phospholipid found in vivo, this model membrane study provides a point of reference for elucidating the role of sterol-lipid interactions in controlling local compositional organization. Our results form the basis for a model that is consistent with cholesterol's ability to modulate the activity of certain neural transmembrane proteins.     

Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.     

Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.     

High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.     

Effects of High Pressure on Glucose Transport in the Human Erythrocyte

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.     

Sweat ammonia excretion during submaximal cycling exercise

This study was undertaken to investigate whether part of the ammonia formed during muscular exercise was excreted with the sweat. Male medical students volunteered for the experiment. They exercised 30 min on a bicycle ergometer at 80 and 40% of the predetermined maximal O2 uptake (VO2max). Exercise at 80% VO2max was performed twice, at room temperature (20 degrees C) and in a cold room (0 degrees C), whereas exercise at 40% was performed only at room temperature (20 degrees C). Blood was collected from the antecubital vein immediately before and after exercise. Sweat was collected from the hypogastric region by use of gauze pads. It was shown that the plasma ammonia level was elevated after exercise at 80% VO2max and remained stable after exercise at 40% VO2max. The volume of sweat produced during exercise at 80% VO2max at 20 degrees C was 428 +/- 138 ml and at 0 degrees C 245 +/- 86 ml and during exercise at 40% VO2max was 183 +/- 69 ml. The ammonia concentration in the sweat after exercise at 80% VO2max at 20 degrees C was 7,140 mumol/l and at 0 degrees C 11,816 mumol/l. After exercise at 40% VO2max, it was 2,076 mumol/l. The total ammonia lost through the sweat during exercise at 80% VO2max was similar at both temperatures, despite the difference in the sweat volume (at 20 degrees C, 3,360 +/- 2,080 mumol; at 0 degrees C, 3,310 +/- 1,250 mumol). During exercise at 40% VO2max, it was 350 +/- 230 mumol. These results show that part of ammonia formed during exercise is lost with sweat. The amount lost increases with increased work rate and the plasma ammonia concentration.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Corticotropin-releasing factor inhibits the acute inflammatory response of rat pawskin to cold injury

The anti-inflammatory effects of human/rat corticotropin-releasing factor (CRF), a 41-residue peptide hormone, on an experimental model of cold injury were examined. Male albino rats were anesthetized with sodium pentobarbital 60 mg/kg ip and the paws immersed for 1 or 2 min in a 22% NaCl solution maintained at -20 +/- 0.5 degrees C. Swelling in response to cold was measured by changes in paw volume using the fluid displacement method, and protein leakages from blood vessels were measured using Evans blue and Monastral blue dyes. Thirty minutes after cold exposure the paw volume increased from 1.5 +/- 0.1 to 2.4 +/- 0.1 ml/paw and the Evans blue content increased from 4 +/- 1 to 178 +/- 9 micrograms/pawskin. These responses to cold were inhibited by 40 to 60% after CRF was injected 56 micrograms/kg sc 30 min before or 28 micrograms/kg iv 10 min before or 5 min after cold exposures. Microscopic studies of the skin showed that CRF reduced leakage of Monastral blue pigment from the vascular compartment into the walls of capillaries and venules. The anti-inflammatory effects of CRF were blocked by alpha-helical CRF(9-41), a CRF receptor antagonist, injected 92 micrograms/kg iv 5 min before and 15 min before cold exposure.     

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.     

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.     

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.