In-situ spectroscopic investigation of ultrasonic assisted unfolding and aggregation of insulin.

It is well-known that fibrillogenesis of proteins can be influenced by diverse external parameters, such as temperature, pressure, agitation or chemical agents. The present preliminary study suggests that ultrasonic excitation at moderate intensities has a significant influence on the unfolding and aggregation behaviour of insulin. Irradiation with an average sound intensity of even as low as 70mW/cm(2) leads to a lowering of the unfolding and aggregation temperature up to 7K. The effect could be explained by an increase of the aggregation kinetics due to ultrasonically induced acoustic micro-streaming in the insulin solution that most probably enhances the aggregation rate. The clear and remarkable effect at relatively low sound intensities offers interesting options for further applications of ultrasound in biophysics and biochemistry. On the other hand, a process that causes a change of kinetics equivalent to 7K also gives a warning signal concerning the safety of those medical ultrasonic devices that work in this intensity range.

The influence of correlated protein-water volume fluctuations on the apparent compressibility of proteins determined by ultrasonic velocimetry.

The elasticity of proteins, expressed by the compressibility, is potentially one of the most important properties of proteins because of the close relationship with its functionality. The compressibility of solutions can be determined by measurements of sound velocity and density. These quantities are related by the Newton-Laplace equation. In order to interpret the apparent compressibility of solutes in highly dilute solutions, it is required to consider the relation between compressibility and sound velocity of the solution using an appropriate reference system. The classical approach usually gives too small values for the apparent compressibility when compared with other methods. We show that the difference can partially be explained if the correlated volume fluctuations of the solvent are taken into consideration. A special attention is given to the compressibility of proteins. Finally, the present paper is not intended to replace established approaches, but it wants to create awareness that the classical mixing rules refer to ideal gasses assuming uncorrelated volume fluctuations and that a considerable part of the hydration effects could be explained by correlated volume fluctuations.

Protein stability and dynamics in the pressure-temperature plane.

The pressure-temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.

High hydrostatic pressure dissociates early aggregates of TTR105-115, but not the mature amyloid fibrils.

A range of disorders such as Alzheimer's disease and type II diabetes have been linked to protein misfolding and aggregation. Transthyretin is an amyloidogenic protein which is involved in familial amyloid polyneuropathy, the most common form of systemic amyloid disease. A peptide fragment of this protein, TTR105-115, has been shown to form well-defined amyloid fibrils in vitro. In this study, the stability of amyloid fibrils towards high hydrostatic pressure has been investigated by Fourier transform infrared spectroscopy. Information on the morphology of the species exposed to high hydrostatic pressure was obtained by atomic force microscopy. The species formed early in the aggregation process were found to be dissociated by relatively low hydrostatic pressure (220 MPa), whereas mature fibrils are pressure insensitive up to 1.3 GPa. The pressure stability of the mature fibrils is consistent with a fibril structure in which there is an extensive hydrogen bond network in a tightly packed environment from which water is excluded. The fact that early aggregates can be dissociated by low pressure suggests, however, that hydrophobic and electrostatic interactions are the dominant factors stabilizing the species formed in the early stages of fibril formation.

The influence of pressure on the lower critical solution temperature miscibility behavior of aqueous solutions of poly(vinyl methyl ether) and the relation to the compositional curvature of the volume of mixing.

In mixtures of PVME and water, the influence of pressure on the LCST miscibility gap is determined covering the whole composition range and pressures from atmospheric pressure up to 900 MPa. The cloud point curve at atmospheric pressure has the characteristic bimodal shape in agreement with literature data. Upon increasing pressure the cloud point curve at the low concentration side decreases with pressure, whereas at the high concentrations the cloud point curve increases with pressure. The overall influence of pressure results in a less pronounced bimodality and ultimately the bimodal shape disappears. In addition to the pressure dependence of the miscibility behavior, the density of mixtures of water and PVME are determined at atmospheric pressure. The experimental excess specific volumes are negative for all measured compositions, but the compositional curvature varies with composition. The curvature of the excess specific volume is positive for the higher concentrations but it is negative in the lower composition range. The density measurements are linked to the pressure dependence of the LCST miscibility behavior using exact thermodynamic relationships. The excess specific volume and miscibility results are shown to be in good agreement. Moreover, it is shown that the Clapeyron equation, which is exact for pure components and also frequently assumed to apply to mixtures, is not valid in the system PVME/water. The system PVME/water is an example where the usual approximation of one-to-one correspondence between curvature and excess volume does not apply. Finally, the molecular origins for the observed excess volume and miscibility behavior are briefly discussed from theoretical and molecular simulation points of view.

The in situ observation of the temperature and pressure stability of recombinant Aspergillus aculeatus pectin methylesterase with Fourier transform IR spectroscopy reveals an unusual pressure stability of beta-helices.

The stability of recombinant Aspergillus aculeatus PME (pectin methylesterase), an enzyme with high beta-helix content, was studied as a function of pressure and temperature. The conformational stability was monitored using FTIR (Fourier transform IR) spectroscopy whereas the functional enzyme stability was monitored by inactivation studies. Protein unfolding followed by amorphous aggregation, which makes the process irreversible, was observed at temperatures above 50 degrees C. This could be correlated to the irreversible enzyme inactivation observed at that temperature. Hydrostatic pressure greater than 1 GPa was necessary to induce changes in the enzyme's secondary structure. No enzyme inactivation was observed at up to 700 MPa. Pressure increased PME stability towards thermal denaturation. At 200 MPa, temperatures above 60 degrees C were necessary to cause significant PME unfolding and loss of activity. These results may be relevant for an understanding of the extreme stability of amyloid fibrils for which beta-helices have been proposed as a structural element.

Single-domain antibody fragments with high conformational stability.

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, beta-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N <--> U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T(m) = 60-80 degrees C), and display high conformational stabilities (DeltaG(H(2)O) = 30-60 kJ mole(-1)). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.

High pressure induces the formation of aggregation-prone states of proteins under reducing conditions.

The pressure stability of ribonuclease A and bovine pancreatic trypsin inhibitor has been investigated with Fourier transform infrared spectroscopy in the presence of the disulfide bond reducing agent 2-mercaptoethanol. The secondary structure of the reduced proteins at high pressure (1 GPa) is not significantly different from the pressure-induced conformation of the native form. Upon decompression under reducing conditions, amorphous aggregates are formed. Such aggregates are not formed upon decompression of the native proteins. Our data demonstrate that high pressure populates, and thus allows the potential characterization of highly aggregation-prone conformations. The relevance of these findings with regard to fibril formation is discussed and the possible role of conformational fluctuations of intermediates on the aggregation pathway is emphasized.

Increased susceptibility of beta-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures.

The stability of beta-glucosidase from the hyperthermophile Pyrococcus furiosus was studied as a function of pressure, temperature and pH. The conformational stability was monitored using FTIR spectroscopy, and the functional enzyme stability was monitored by inactivation studies. The enzyme proved to be highly piezostable and thermostable, with an unfolding pressure of 800 MPa at 85 degrees C. The tentative pressure-temperature stability diagram indicates that this enzyme is stabilized against thermal unfolding at low pressures. The activity measurements showed a two-step inactivation mechanism due to pressure that was most pronounced at lower temperatures. The first part of this inactivation took place at pressures below 300 MPa and was not visible as a conformational transition. The second transition in activity was concomitant with the conformational transition. An increase in pH from 5.5 to 6.5 was found to have a stabilizing effect.

Protein unfolding, amyloid fibril formation and configurational energy landscapes under high pressure conditions.

High hydrostatic pressure induces conformational changes in proteins ranging from compression of the molecules to loss of native structure. In this tutorial review we describe how the interplay between the volume change and the compressibility leads to pressure-induced unfolding of proteins and dissociation of amyloid fibrils. We also discuss the effect of pressure on protein folding and free energy landscapes. From a molecular viewpoint, pressure effects can be rationalised in terms of packing and hydration of proteins.

The sound velocity in ideal liquid mixtures from thermal volume fluctuations.

The selection of the correct mixing rule for sound velocity in ideal liquid mixtures determines the interpretation of the sound velocity in real mixtures. This is especially important for the determination of apparent properties of solutes, such as their apparent compressibility. There are different approaches reported in the literature, and this article presents a new derivation of the mixing rule based on statistical mechanics. It is shown that the correlation of volume fluctuations between adjacent components has a crucial influence on the ideal mixing rule.

Pressure-induced protein unfolding in the ternary system AOT-octane-water is different from that in bulk water.

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of alpha-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, alpha-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.

Temperature-induced dissociation of protein aggregates: accessing the denatured state.

The thermal denaturation of lysozyme and ribonuclease A (RNase A) under reducing and nonreducing conditions at neutral pH has been monitored by Fourier transform infrared spectroscopy. In the absence of the reductant, lysozyme and RNase A undergo apparent three- and two-state denaturation, respectively, as observed from the conformation-sensitive amide I' band. For both proteins the hydrogen-deuterium exchange takes place at lower temperatures than the main denaturation temperatures, suggesting that a transient denaturation mechanism occurs. The observed transition at 51.2 degrees C during the denaturation of lysozyme is attributed to this transient effect, rather than to the loss of tertiary structure. Under reducing conditions lysozyme aggregates during the heating phase, whereas RNase A shows only a minor aggregation, which further increases during the cooling step. The reduced stability of both proteins can be correlated with the transient denaturation behavior, which is also suggested to be involved in protein aggregation at physiologically relevant temperatures. In addition, it is shown that when the temperature is further increased, the amorphous aggregates dissociate. Comparison of the dissociated states with the denatured states obtained under nonreducing conditions indicates that these states have the same conformation. By using a two-dimensional correlation analysis we were able to show that the dissociation is preceded by a conformational change. It is argued that this extends to other types of perturbation.

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin.

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study.

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.

Pressure- and temperature-induced unfolding and aggregation of recombinant human interferon-gamma: a Fourier transform infrared spectroscopy study.

The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-gamma (rhIFN-gamma) and its biologically inactive truncated form rhIFN-Delta C15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the alpha-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5 M MgCl(2) significantly reduces the pressure stability. Releasing the pressure below 300 MPa results in the formation of intermolecular antiparallel beta-sheets, which is seldom observed. This suggests that the intermolecular beta-sheet of rhIFN-gamma is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of alpha-helical structure into intermolecular antiparallel beta-sheet, as is usually observed for most proteins.

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.

Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH).

The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.