László Smeller

Protein structure and dynamics at high pressure

The effect of pressure on the structure and dynamics of proteins is discussed in the framework of the pressure-temperature stability phase diagram. The elastic (reversible) properties, thermal expansion, compressibility and heat capacity, are correlated with the entropy, volume, and the coupling between entropy and volume fluctuations respectively. The experimental approaches that can be used to measure these quantities are reviewed. The plastic (conformational) changes reflect the changes in these properties in the cold, pressure and heat denaturation.

Pressure-temperature phase diagrams of biomolecules

The pressure-temperature phase diagram of various biomolecules is reviewed. Special attention is focused on the elliptic phase diagram of proteins. The phenomenological thermodynamic theory describing this diagram explains the heat, cold and pressure denaturations in a unified picture. The limitations and possible developments of this theory are discussed as well. It is pointed out that a more complex diagram can be obtained when the intermolecular interactions are also taken into account. In this case metastable states appear on the pressure-temperature (p-T) diagram due to intermolecular interactions. Pressure-temperature phase diagrams of other biopolymers are also discussed. While the p-T diagrams of helix-coil transition of nucleic acids and of gel-liquid crystal transition of lipid bilayers are non-elliptical, those of gelatinization of starch and of phase separation of some synthetic polymers show an elliptic profile, similar to that of proteins. Finally, the p-T diagram of bacterial inactivation is shown to be elliptic. From the point of view of basic science, this fact shows that the key factor of inactivation should be the protein type, and from the viewpoint of practical applications, it serves as the theoretical basis of pressure treatment of biosystems.

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).

2D FT-IR spectroscopy analysis of the pressure-induced changes in proteins

Abstract In this paper, we apply for the first time two-dimensional (2D) correlation spectroscopy to analyze pressure-induced changes in proteins. We show that it is possible to distinguish between hydrogen-deuterium (H/D) exchange and conformational changes from the synchronous and the asynchronous spectra. From the sequence of the spectral changes it can be concluded that the initial partial unfolding of the secondary structure enhances the exchange, which is followed by further conformational changes. The exchange process is complete below ca. 0.4 GPa. The elastic distortions of the protein at higher pressures seem not to be accompanied by H/D exchange.

How to Minimize Certain Artifacts in Fourier Self-Deconvolution

Computers are common tools in spectroscopy and they offer new possibilities for extracting useful information from experimental data. One of these tools is resolution enhancement of infrared spectra with Fourier self-deconvolution. This procedure is often used for the analysis of the amide I band in proteins. It is part of most commercial spectroscopic software packages, especially in the FT-IR software in which Fourier transformation is the basis of the experimental procedure. Thus Fourier self-deconvolution is becoming a powerful tool in spectroscopy. Since the number of users is expected to increase rapidly, a warning against the problems and pitfalls of the technique is appropriate. Usually one starts the software with a certain set of deconvolution parameters and varies them by a visual check of the result. The resolution enhancement is increased until the spectrum looks unrealistic. Two kinds of artifacts can occur during the process: over-deconvolution, which results in the appearance of side lobes, and the uncontrolled increase of the noise.

High pressure effects on allergen food proteins.

There are several proteins, which can cause allergic reaction if they are inhaled or ingested. Our everyday food can also contain such proteins. Food allergy is an IgE-mediated immune disorder, a growing health problem of great public concern. High pressure is known to affect the structure of proteins; typically few hundred MPa pressure can lead to denaturation. That is why several trials have been performed to alter the structure of the allergen proteins by high pressure, in order to reduce its allergenicity. Studies have been performed both on simple protein solutions and on complex food systems. Here we review those allergens which have been investigated under or after high pressure treatment by methods capable of detecting changes in the secondary and tertiary structure of the proteins. We focus on those allergenic proteins, whose structural changes were investigated by spectroscopic methods under pressure in correlation with the observed allergenicity (IgE binding) changes. According to this criterion we selected the following allergen proteins: Mal d 1 and Mal d 3 (apple), Bos d 5 (milk), Dau c 1 (carrot), Gal d 2 (egg), Ara h 2 and Ara h 6 (peanut), and Gad m 1 (cod).

Determination of the secondary structure of proteins at high pressure

Abstract A combined deconvolution and multiband fitting method is proposed for the determination of the effect of high pressure on the secondary structure of proteins in solution and in membranes with Fourier transform infrared spectroscopy. The method allows the determination of the pressure-induced elastic effect, due to the compression of the hydrogen bonds, as well as the conformational changes of the secondary structures. The effect of pressure on gramicidin A is shown as an example.

Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathways.

As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full‐length recombinant PrP undergoes β‐sheet aggregation on high‐pressure‐induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, AggHP, which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, AggHP transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially β‐sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, IHP, is formed under high pressure. After pressure release, IHP relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, IHP, constitutes a necessary prerequisite for the formation of amyloids. The use of high‐pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.

The enzyme horseradish peroxidase is less compressible at higher pressures.

Fluorescence line-narrowing (FLN) spectroscopy at 10 K was used to study the effect of high pressure through the prosthetic group in horseradish peroxidase (HRP), which was Mg-mesoporphyrin (MgMP) replacing the heme of the enzyme. The same measurement was performed on MgMP in a solid-state amorphous organic matrix, dimethyl sulfoxide (DMSO). Series of FLN spectra were registered to determine the (0, 0) band shape through the inhomogeneous distribution function (IDF). In the range of 0-2 GPa a red-shift of the IDF was determined, and yielded the isothermal compressibility of MgMP-HRP as 0.066 GPa(-1), which is significantly smaller than that found earlier as 0.106 GPa(-1) by fine-tuning the pressure in the range up to 1.1 MPa. The vibrational frequencies also shifted with pressure increase, as expected. The compressibility in the DMSO matrix was smaller, 0.042 GPa(-1), both when the pressure was applied at room temperature before cooling to 10 K, or at 10 K. At 200 K or above, the bimodal (0, 0) band shape in DMSO showed a population conversion under pressure that was not observed at or below 150 K. A significant atomic rearrangement was estimated from the volume change, 3.3 +/- 0.7 cm(3)/mol upon conversion. The compressibility in proteins and in amorphous solids seems not to significantly depend on the temperature and in the protein it decreases toward higher pressures.

Pressure tuning spectroscopy of the low‐frequency Raman spectrum of liquid amides

The effect of hydrostatic pressure up to 4 kbar on the low frequency Raman spectrum of hydrogen bonded (formamide, N‐methylformamide, N‐ethylformamide, and N‐methylpropionamide) and nonhydrogen bonded (N,N’‐dimethylformamide and N,N’‐dimethylacetamide) liquid amides is reported. A shift of ca. 4.0 cm−1/kbar is observed for the nonhydrogen bonded amides. For the hydrogen bonded amides the shift is ca. 2.0–3.0 cm−1/kbar. The difference may be explained by the compression of the hydrogen bond which affects the librational motion of the molecules.