Temperature and pressure dependence of protein stability: the engineered fluorescein-binding lipocalin FluA shows an elliptic phase diagram.

We have measured the equilibrium constant for the denaturation transition of the engineered fluorescein-binding lipocalin FluA as a function of pressure and temperature, taking advantage of the fact that the ligand's fluorescence is almost fully quenched when complexed with the folded protein, but reversibly reappears on denaturation. From the equilibrium constant as a function of pressure and temperature all of the involved thermodynamic parameters of protein folding, in particular the changes in entropy and volume, compressibility, thermal expansion, and specific heat, were deduced in a global fitting procedure. Assuming that these parameters are independent of temperature and pressure, we can demonstrate from the ratio of Deltabeta, Deltaalpha(2), DeltaC(p) that the phase diagram of protein folding assumes an elliptic shape. Furthermore, we can show that the thermodynamic condition for such an elliptic phase diagram is related to the degree of correlation between the fluctuations of the changes in volume and enthalpy at the phase boundary. For the protein investigated this correlation is low, as generally expected for highly degenerate systems. Our study suggests that the elliptic phase diagram is a consequence of the inherent conformational disorder of proteins and that it may be viewed as the thermodynamic manifestation of the high degeneracy of conformational energies that is characteristic for this class of macromolecules.

Terrylene in hexadecane revisited: a hole burning study.

Holes burnt into the absorption spectrum of terrylene in hexadecane have quite unusual features: spectral diffusion behavior under thermal cycles shows a narrowing regime at very low temperatures (2-5 K) followed by a plateau region (up to about 13 K) and a broadening regime (T>13 K). Thermal line broadening (quasihomogeneous linewidth) shows a nonmonotonous behavior as a function of temperature: at around 4 K there is a maximum followed by a flat minimum and the onset of strong broadening at higher temperatures. Finally, the central hole shows one-sided narrowly spaced side features. This behavior is interpreted within the frame of a two-site model. One of the two sites can be well described by a standard two level system; the other, however, shows characteristic features of a multilevel system. The two sites are characterized by strongly different optical linewidths, phototransformation yields, and thermal stabilities.

Protein elasticity determined by pressure tuning of the tyrosine residue of ubiquitin.

We determined the isotropic, isothermal compressibility of ubiquitin by pressure tuning spectral holes burnt into the red edge of the absorption spectrum of the single tyrosine residue. The pressure shift is perfectly linear with burn frequency. From these data, a compressibility of 0.086 GPa(-1) in the local environment of the tyrosine residue could be determined. This value fits nicely into the range known for proteins. Although the elastic behavior at low temperatures does not show any unusual features, the pressure tuning behavior at room temperature is quite surprising: the pressure-induced spectral shift is close to zero, even up to very high pressure levels of 0.88 GPa, well beyond the denaturation point. The reason for this behavior is attributed to equally strong blue as well as red spectral pressure shifts resulting in an average pressure-induced solvent shift that is close to zero.

Investigation of spectral diffusion in ribonuclease by photolabeling of intrinsic aromatic amino acids.

Spectral diffusion dynamics in ribonuclease A was observed via the broadening of photochemical holes burned into the absorption spectrum of intrinsic tyrosine residues. Unlike previous results based on hole burning of chromophores in the pockets of heme proteins, where spectral diffusion develops according to a power law in time, the dynamics in ribonuclease follow a logarithmic law. The results suggest that the experiment preferentially labels the tyrosines located on the surface of the protein where the two-level system dynamics of the glass host matrix exert a strong influence.

Thermal-cycling-induced spectral diffusion and thermal barriers in anisole-doped cyclohexane, an unusual multiphase host-guest system.

The host-guest system of anisole incorporated into a cyclohexane matrix was investigated in a series of hole-burning experiments. This system is unusual in that cyclohexane can freeze into coexisting solid phases. The hole-burning experiments support the existence of two crystalline phases and one disordered phase. A second surprising characteristic of this system is that the quasi-line absorption features of the spectra appear inverted at low temperature because of unexpected dominance of fluorescence and phosphorescence.

Hole burning spectroscopy of ribonuclease A.

We present pressure tuning hole burning experiments with the enzyme ribonuclease A using the UV-absorbing amino acid tyrosine as a probe. We show that, at 2 K, the protein is intact, and that at least four different regions which we associate with different tyrosine sites can be distinguished through their specific response to pressure. For one site we could determine the compressibility to 0.15 GPa(-1). Upon denaturing the protein with guanidine hydrochloride, one of the tyrosine sites is preserved to a large extent. Reducing the sulfur bonds has a more drastic effect: the tyrosine sites lose most of their individual features and their compressibilities come close to that of tyrosine in solution.

Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM.

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed.

Stability of proteins: temperature, pressure and the role of the solvent.

We focus on the various aspects of the physics related to the stability of proteins. We review the pure thermodynamic aspects of the response of a protein to pressure and temperature variations and discuss the respective stability phase diagram. We relate the experimentally observed shape of this diagram to the low degree of correlation between the fluctuations of enthalpy and volume changes associated with the folding-denaturing transition and draw attention to the fact that one order parameter is not enough to characterize the transition. We discuss in detail microscopic aspects of the various contributions to the free energy gap of proteins and put emphasis on how a cosolvent may either enlarge or diminish this gap. We review briefly the various experimental approaches to measure changes in protein stability induced by cosolvents, denaturants, but also by pressure and temperature. Finally, we discuss in detail our own molecular dynamics simulations on cytochrome c and show what happens under high pressure, how glycerol influences structure and volume fluctuations, and how all this compares with experiments.

Local compressibilities of proteins: comparison of optical experiments and simulations for horse heart cytochrome-c.

Spectroscopy with probe molecules yields local information on the environment of the probe. In this article we compare local compressibilities of cytochrome-c as obtained from molecular dynamics simulations with experimental results as obtained from spectroscopic measurements. The simulations show that the protein-core around the heme is much less compressible in a glycerol/water solvent than in pure water. The pocket is also much less compressible than the protein as a whole, although the compressibility of the water inside the rather incompressible protein-core is almost liquidlike. We show that the local compressibility values capture the collective correlations of local volume fluctuations with volume fluctuations in the surrounding protein-solvent system. The decoupling of the volume fluctuations of the core from the solvent shell explains the reduction of the heme-core-compressibility in glycerol/water solvent. This decoupling could be traced back to the suppression of the exchange between pocket-water and hydration-shell-water upon addition of glycerol as co-solvent.

Local compressibilities in insulin as determined from pressure tuning hole burning experiments and MD simulations.

We present a hole burning study on insulin in a glycerol-water solvent by using the intrinsic amino acid tyrosine as a photochemical probe. The focus of the experiments is on the comparative pressure response of the spectral holes for insulin in its native state, in its chemically denatured state and for tyrosine in the glycerol-water solvent. From an analysis of the color effect of the pressure response, we can identify two different spectral ranges characterized by a markedly different sensitivity to pressure. We conclude that at least two tyrosines (or two groups of tyrosines) out of the eight in the insulin dimer are photochemically labeled, and that they are characterized by markedly different compressibilities, namely 0.08 and 0.13 GPa(-1), respectively. An interesting observation concerns the compressibility in the unfolded state: It is significantly lower and closer to the value measured for the pure tyrosine molecule in a glycerol-water solvent. In contrast to the native state, the response of the various tyrosines in the unfolded state to pressure variations is quite uniform. The experiments are compared with MD simulations of monomeric insulin at ambient temperature. The computational results show that the local compressibilities around the different tyrosines vary significantly and that they strongly depend on whether just the first shell of molecules or the first two shells are included in the local volume.

Protein phase diagrams: the physics behind their elliptic shape.

We relate the condition for the elliptic shape of the phase diagram of proteins to the degree of correlation in the fluctuations of the changes of enthalpy and volume at the denaturing-refolding transition. Since this degree cannot be larger than 1, hyperbolically shaped diagrams are not likely to exist. Experiments show that the correlation factor is actually quite low for proteins implying that one-order parameter is not enough to describe the folding-denaturing transition. These findings seem to be the thermodynamic manifestation of the glasslike properties of proteins despite the fact that the transition itself is of first order.

Aging dynamics in globular proteins: summary and analysis of experimental results and simulation by a modified trap model.

Recent results of spectral diffusion experiments by spectral hole-burning techniques carried out at cryogenic temperatures on various monomeric heme proteins unequivocally show interesting new features of conformational dynamics of globular proteins that were not emphasized in the literature until now. These new aspects of the protein dynamics are anomalous diffusion and the aging effect. Here, using the similarities between proteins and glassy systems, we present a model which can interpret the line broadening and-through this effect-the aging phenomenon as well. Leaving untouched the widely accepted energy landscape (EL) concept for the general description of protein dynamics, we concentrate on the bottom of the funnel-like EL, because this part corresponds to the native state(s) at low temperature. We suggest that the overall shape of the EL at the lowest energy range is rather smooth, but on a finer scale it consists of traps. The dynamics is defined by sequential jumps among these traps and the process is described by a Master equation, where the hopping rate only depends on the parameters of the starting state. This model was adapted to interpret the common results of spectral diffusion experiments. We tested our model in the simplest case by computer simulation, and it shows excellent agreement with the experimental data. To our knowledge this is the first work where a theoretical interpretation of the aging dynamics of proteins is directly and quantitatively related to the experimental observations. We also show that the model, after the generalization that the traps are hierarchically organized, is in accordance with the concept of other well-known EL models.

Labeling proteins via hole burning of their aromatic amino acids: pressure tuning spectroscopy of BPTI.

We demonstrate hole burning on a protein by using an intrinsic aromatic amino acid as a probe. The protein is bovine pancreatic trypsin inhibitor (BPTI), the labeled amino acid is tyrosine. Only one of the four tyrosines could be burned. As an application we present pressure tuning experiments from which the local compressibility around the burned tyrosine probe is determined.

Stability diagram and unfolding of a modified cytochrome c: what happens in the transformation regime?

We determined the stability diagram of a modified cytochrome c protein in a glycerol water mixture by measuring the first and the second moment of the fluorescence from the chromophore as a function of temperature and pressure. Temperature and pressure were varied between 273 and 363 K and 0.0001 and 1 GPa, respectively. The shift of the fluorescence maximum showed a characteristic sigmoid-like pattern from which information on the microscopic processes during unfolding is obtained: as the transformation regime is entered, the fluorescence shows a significant blue shift. The conclusion is that water molecules get into contact with the chromophore. They lead to strong electrostatic contributions in the solvent shift, which counteract the red shifting dispersion interactions. Assuming that there are just two relevant states that determine the stability diagram, the complete set of thermodynamic parameters can be determined from the data. However, under certain pressure-temperature conditions the fluorescence pattern is more complicated, pointing toward reentrant transitions and, possibly, to consecutive steps in the unfolding process.