Fourth derivative UV-spectroscopy of proteins under high pressure II. Application to reversible structural changes

The structural basis and the thermodynamics of pressure induced reversible spectral transitions in the fourth derivative ultraviolet absorbance spectra of proteins were analysed as described in the preceding paper. Three proteins were studied: adrenodoxin (a small iron-sulphur protein that serves as an electron donor for cytochrome P450scc), ribonuclease A, and methanol dehydrogenase (a tetrameric protein). Fourth derivative spectroscopy is used to probe important mechanistic aspects of these proteins. For adrenodoxin, the results suggest that one or two phenylalanines interact with the iron-sulphur redox centre. High pressure denaturation of ribonuclease leads to a molten globule like structure that also occurs as an intermediate in the high temperature induced denaturation process. This state is characterised by the local dielectric constant in the vicinity of tyrosines. Methanol dehydrogenase was found to be very stable towards pressure. High pressure appears to strengthen the interaction between the two -subunits possibly through the increased interaction of four tryptophans with other aromatic amino acids.     

Fourth derivative UV-spectroscopy of proteins under high pressure I. Factors affecting the fourth derivative spectrum of the aromatic amino acids

A tunable fourth derivative UV absorbance method based on a variable spectral shift has been developed and compared to the Savitzky-Golay method and the analytical derivative. The parameters of the method were optimised for the analysis of the UV absorbance spectra of the aromatic amino acids to quantify the effect of decreasing solvent polarity on their fourth derivative spectra. The wavelength of the highest maximum (_max) (for tyrosine and phenylalanine) or the amplitude of the highest maximum (A_max) (for tryptophan), were shown to depend linearly on the dielectric constant of the solvent, ranging from water to cyclohexane. The only effect of pressure in the 1 to 500 MPa range is a small decrease in the fourth derivative amplitude. This method appears therefore as a suitable tool to evaluate changes of the dielectric constant in the vicinity of the aromatic amino acids in proteins which undergo pressure induced structural changes.     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

A new reference material for UV-visible circular dichroism spectroscopy

To obtain accurate and consistent measurements from circular dichroism (CD) instruments over time and from different laboratories, it is important that they are properly calibrated. The characteristics of the available reference materials are not ideal to ensure proper calibration as they typically only give peaks in one or two spectral regions, and often have issues concerning purity and stability. Currently either camphor sulfonic acid or ammonium camphor sulfonate are used. The latter can be an unstable, slightly hygroscopic secondary standard compound with only one characterized CD band. The former is the very hygroscopic primary standard for which only one enantiomer is readily available. We have synthesized a new reference material for CD, Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid) which addresses these problems. It is extremely stable and available in both enantiomeric forms. The CD spectrum of Na[Co(EDDS)].H(2)O has nine distinct peaks between 180 and 599 nm. It thus fulfils the principal requirements for CD calibration chemical standards and has the potential to be used to ensure good practice in the measurement of CD data, providing two spectra of equal magnitude and opposite sign for a given concentration and path length. We have carried out an interlaboratory comparison using this material and show how it can be used to improve CD comparability between laboratories. A fitting algorithm has been developed to assess CD spectropolarimeter performance between 750 and 178 nm. This could be the basis of a formal quality control process once criteria for performance have been decided.     

Starch gelatinization under pressure studied by high pressure DSC

The gelatinization process of waxy corn starch under different pressures up to 10.0 MPa was investigated using a high pressure DSC. Compressed air and carbon dioxide were used as pressure resources. Effect of pressure and annealing under pressure on gelatinization of waxy corn starch was systematically studied, in particular on the gelatinization temperature and enthalpy. The results show that the peak temperature of gelatinization was increased slightly initially then remained stable with increasing pressure. The gelatinization enthalpy was decreased under pressure processing. Annealing the starch under pressure condition, just below its gelatinization temperature, increased gelatinization temperature but kept gelatinized enthalpy constant. Morphologies of starch granules treated under pressure were studied using an optical microscope and SEM. There is no discernable difference of starch granules treated with and without pressure, which indicates the pressures are not high enough to destroy crystalline structure. The intensity of the pressure acts as a key factor to influence the gelatinization of starch rather than the nature of the gas. Effect of pressure on the multi-endotherm detected by DSC for starch with intermediate water is used to study the mechanisms. The effect of pressure can be explained by the enhancement of water diffusion in the amorphous range.     

Cold denaturation of proteins under high pressure

The advantageous usage of the high pressure technique in studies of cold denaturation of proteins is reviewed, with a brief explanation of the theoretical background of this universal phenomenon. Various experimental results are presented and discussed, explaining the plausible image of the cold denatured state of proteins. In order to understand more clearly this phenomenon and protein structure transition in general, several studies on model polymer systems are also reviewed.     

Monitoring microbial redox transformations of metal and metalloid elements under high pressure using in situ X-ray absorption spectroscopy

X-ray absorption spectroscopy is a well-established method for probing local structural and electronic atomic environments in a variety of systems. We used X-ray absorption near-edge structure (XANES) spectroscopy for monitoring in real-time conditions selenium reduction in situ in live cultures of Shewanella oneidensis MR-1 under high hydrostatic pressure. High-quality XANES data show that Shewanella oneidensis MR-1 reduces selenite Se(IV) to red elemental selenium Se(0) up to 150 MPa without any intermediate redox state. MR-1 reduces all selenite provided (5-10 mM) between 0.1 and 60 MPa. Above 60 MPa the selenite reduction yield decreases linearly with pressure and the activity is calculated to stop at 155 ± 5 MPa. The analysis of cultures recovered after in situ measurements showed that the decrease in activity is linked to a decrease in viability. This study emphasizes the promising potential of XANES spectroscopy for real-time probing in situ microbial redox transformations of a broad range of metal and metalloid elements in live samples, including under high hydrostatic pressure.     

Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.     

Bilayer phase transitions of N-methylated dioleoylphosphatidylethanolamines under high pressure

The bilayer phase transitions of four kinds of unsaturated phospholipids with different-sized polar head groups, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-N-methylethanolamine (DOMePE), dioleoylphosphatidyl-N,N-dimethylethanolamine (DOMe2PE) and dioleoylphosphatidylcholine (DOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. DSC thermogram and light-transmittance curve for each phospholipid vesicle solution exhibited only one phase transition under ambient pressure, respectively. The light-transmittance of DOPC solution at pressure higher than 234 MPa abruptly increased stepwise at two temperatures, which corresponds to the appearance of stable subgel and lamellar gel phases under high pressure in addition to the liquid crystal phase. The constructed temperature (T)-pressure (p) phase diagrams were compared among these phospholipids. The phase-transition temperatures of the phospholipids decreased stepwise by N-methylation of the head group. The slops of the T-p phase boundary (dT/dp) of DOPE, DOMePE and DOMe2PE bilayers (0.127-0.145 K MPa-1) were found to be close to that of the transition from the lamellar crystal (or subgel; Lc) phase to the liquid crystal (Lalpha) phase for DOPC bilayer (0.131 K MPa-1). On the other hand, the dT/dp value of the main transition from the lamellar gel (Lbeta) phase to the Lalpha phase for DOPC bilayer (0.233 K MPa-1) was significantly different from that of the Lc/Lalpha transition, hence both curves intersected with each other at 234 MPa. The thermodynamic quantities associated with the phase transition of DOPE, DOMePE and DOMe2PE bilayers had also similar values to those for the Lc/Lalpha transition of DOPC bilayer. Taking into account of the values of transition temperature, dT/dp and thermodynamic quantities compared with the corresponding results of saturated phospholipids, we identified the phase transitions observed in the DOPE, DOMePE and DOMe2PE bilayers as the transition from the Lc phase to the Lalpha phase although they have been regarded as the main transition in the previous studies. The Lbeta phase is probably unstable for DOPE, DOMePE and DOMe2PE bilayers at all pressures, it exists as a metastable phase at pressures below 234 MPa while as a stable phase at pressures above 234 MPa in DOPC bilayer. The difference in phase stability among the phospholipid bilayers is originated from that in hydration structure of the polar head groups.     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.     

Electron transfer and electronic energy relaxation under high hydrostatic pressure

The following question has been addressed in the present work. How external high (up to 8 kbar) hydrostatic pressure acts on photoinduced intramolecular electron transfer and on exciton relaxation processes? Unlike phenomena, as they are, have been studied in different systems: electron transfer in an artificial Zn-porphyrin-pyromellitimide (ZnP-PM) supramolecular electron donor-acceptor complex dissolved in toluene measured at room temperature; exciton relaxation in a natural photosynthetic antenna protein called FMO protein measured at low temperatures, between 4 and 100 K. Spectrally selective picosecond time-resolved emission technique has been used to detect pressure-induced changes in the systems. The following conclusions have been drawn from the electron transfer study: (i) External pressure may serve as a potential and sensitive tool not only to study, but also to control and tune elementary chemical reactions in solvents; (ii) Depending on the system parameters, pressure can both accelerate and inhibit electron transfer reactions; (iii) If competing pathways of the reaction are available, pressure can probably change the branching ratio between the pathways; (iv) The classical nonadiabatic electron transfer theory describes well the phenomena in the ZnP-PM complex, assuming that the driving force or/and reorganisation energy depend linearly on pressure; (v) A decrease in the ZnP-PM donor-acceptor distance under pressure exerts a minor effect on the electron transfer rate. The effect of pressure on the FMO protein exciton relaxation dynamics at low temperatures has been found marginal. This may probably be explained by a unique structure of the protein [D.E. Trondrud, M.F. Schmid, B.W. Matthews, J. Mol. Biol. 188 (1986) p. 443; Y.-F. Li, W. Zhou, E. Blankenship, J.P. Allen, J. Mol. Biol., submitted]. A barrel made of low compressibility beta-sheets may, like a diving bell, effectively screen internal bacteriochlorophyll a molecules from external influence of high pressure. The origin of the observed slow pico = and subnanosecond dynamics of the excitons at the exciton band bottom remains open. The phenomenon may be due to weak coupling of phonons to the exciton states or/and to low density of the relevant low-frequency ( approximately 50 cm(-1)) phonons. Exciton solvation in the surrounding protein and water-glycerol matrix may also contribute to this effect. Drastic changes of spectral, kinetic and dynamic properties have been observed due to protein denaturation, if the protein was compressed at room temperature and then cooled down, as compared to the samples, first cooled and then pressurised.     

Second derivative fluorescence spectroscopy of tryptophan in proteins

The second derivatives of N-acetyl- -tryptophan amide (AcTrpNH₂) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH₂ fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase–PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.     

High pressure microscopy--a powerful tool for monitoring cells and macromolecules under high hydrostatic pressure.

A high pressure chamber, which withstands a pressure up to 300 MPa has been developed. The so-called HPDS (Hartmann, Pfeifer, Dornheim, Sommer) High Pressure Cell in combination with an inverted microscope and an analysis system allows brilliant microscopic colour pictures with an optical resolution better than 0.56 microm. The pressure chamber allows the in situ observation of dynamic changes of microscopic structures in bright field, phase contrast and fluorescence microscopy. This publication should demonstrate the capabilities of the system using results of experiments with two types of Spirogyra algae. The pictures have shown significant variations of the chloroplasma and the cell wall membrane at pressures of up to 120 MPa. The new system provides a simple way to perform microscopic analyses at pressures of up to 300 MPa.