Helén Jansson

A unified model of protein dynamics

Protein functions require conformational motions. We show here that the dominant conformational motions are slaved by the hydration shell and the bulk solvent. The protein contributes the structure necessary for function. We formulate a model that is based on experiments, insights from the physics of glass-forming liquids, and the concepts of a hierarchically organized energy landscape. To explore the effect of external fluctuations on protein dynamics, we measure the fluctuations in the bulk solvent and the hydration shell with broadband dielectric spectroscopy and compare them with internal fluctuations measured with the Mössbauer effect and neutron scattering. The result is clear. Large-scale protein motions are slaved to the fluctuations in the bulk solvent. They are controlled by the solvent viscosity, and are absent in a solid environment. Internal protein motions are slaved to the beta fluctuations of the hydration shell, are controlled by hydration, and are absent in a dehydrated protein. The model quantitatively predicts the rapid increase of the mean-square displacement above ≈200 K, shows that the external beta fluctuations determine the temperature- and time-dependence of the passage of carbon monoxide through myoglobin, and explains the nonexponential time dependence of the protein relaxation after photodissociation.

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water-glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T(g) is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the alpha-relaxation of the main water relaxation, although it does not contribute to the calorimetric T(g). This is in contrast to myoglobin in water-glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.

Role of solvent for the dynamics and the glass transition of proteins.

For the first time, a systematic investigation of the glass transition and its related dynamics of myoglobin in water-glycerol solvent mixtures of different water contents is presented. By a combination of broadband dielectric spectroscopy and differential scanning calorimetry (DSC), we have studied the relation between the protein and solvent dynamics with the aim to better understand the calorimetric glass transition, T(g), of proteins and the role of solvent for protein dynamics. The results show that both the viscosity related α-relaxation in the solvent as well as several different protein relaxations are involved in the calorimetric glass transition, and that the broadness (ΔT(g)) of the transition depends strongly on the total amount of solvent. The main reason for this seems to be that the protein relaxation processes become more separated in time with decreasing solvent level. The results are compared to that of hydrated myoglobin where the hydration water does not give any direct contribution to the calorimetric T(g). However, the large-scale α-like relaxation in the hydration water is still responsible for the protein dynamics that freeze-in at T(g). Finally, the dielectric data show clearly that the protein relaxation processes exhibit similar temperature dependences as the α-relaxation in the solvent, as suggested for solvent-slaved protein motions.

Dynamics of water in a molecular sieve by quasielastic neutron scattering

We have investigated the dynamics of water confined in a molecular sieve, with a cylindrical pore diameter of 10 A, by means of quasielastic neutron scattering (QENS). Both the incoherent and coherent intermediate scattering functions I(Q,t) were determined by time-of-flight QENS and the neutron spin-echo technique, respectively. The results show that I(Q,t) is considerably more stretched in time with a slightly larger average relaxation time in the case of coherent scattering. From the Q dependence of I(Q,t) it is clear that the observed dynamics is almost of an ordinary translational nature. A comparison with previous dielectric measurements suggests a possible merging of the alpha and beta relaxations of the confined water at T=185 K, although the alpha relaxation cannot be directly observed at lower temperatures due to the severe confinement. The present results are discussed in relation to previous results for water confined in a Na-vermiculite clay, where the average relaxation time from spin-echo measurements was found to be slower than in the present system (particularly at low temperatures).

Dynamics of a protein and its surrounding environment: A quasielastic neutron scattering study of myoglobin in water and glycerol mixtures

In this quasielastic neutron scattering (QENS) study we have investigated the relation between protein and solvent dynamics. Myoglobin in different water:glycerol mixtures has been studied in the temperature range of 260-320 K. In order to distinguish between solvent and protein dynamics we have measured protonated as well as partly deuterated samples. As commonly observed for bulk as well as for confined water, the dynamics of the surrounding solvent is well described by a jump diffusion model. The intermediate scattering function I(Q,t) from the protein (partly deuterated samples) was analyzed by fitting a single Kohlrausch-Williams-Watts (KWW) stretched exponential function to the data. However, due to the limited experimental time window, two different curve fitting approaches were used. The first one was performed with the assumption that I(Q,t) decays to zero at long times, i.e., it was assumed that all protein relaxations that are observed on the experimental time scale, as well as would be observed on longer time scales, can be described by a single KWW function. In the second approach we instead assumed that both the protein relaxation time tau(p) and the stretching parameter beta(KWW) were Q-independent, i.e., we assumed that the protein dynamics is dominated by more local motions. Advantages and disadvantages of both approaches are discussed. The first approach appears to work best at higher Q-values, indicating a power law relation of the Q-dependent protein dynamics for all samples and temperatures, whereas the second approach seems to work at lower Q-values, where the expected confined diffusion of hydrogen atoms in the protein gives the assumed Q-independent relaxation time. Independent of the chosen approach we find a significant correlation between the average relaxation time of the protein and the diffusion constant (or in this case the related relaxation time) of the solvent. However, the correlation is not perfect since the average relaxation time of the protein is more strongly dependent on the total amount of solvent than the diffusion constant of the solvent itself. Thus, the average relaxation time of the protein decreases not only with increasing solvent mobility, but also with increasing solvent content.

The Role of Trehalose for the Stabilization of Proteins.

Understanding of how the stabilization mechanism of trehalose operates on biological molecules against different types of environmental stress could prove to gain great advancements in many different types of conservation techniques, such as cryopreservation or freeze-drying. Many theories exist that aim to explain why trehalose possesses an extraordinary ability to stabilize biomolecules. However, all of them just explain parts of its mechanism and a comprehensive picture is still lacking. In this study, we have used differential scanning calorimetry (DSC) and viscometry measurements to determine how the glass transition temperature Tg, the protein denaturation temperature Tden, and the dynamic viscosity depend on both the trehalose and the protein concentration in myoglobin-trehalose-water systems. The aim has been to determine whether these physical properties are related and to gain indirect structural insights from the limits of water crystallization at different concentration ratios. The results show that for systems without partial crystallization of water the addition of protein increases Tg, most likely due to the fact that the protein adsorbs water and thereby reduces the water content in the trehalose-water matrix. Furthermore, these systems are generally decreasing in Tden with an increasing protein concentration, and thereby also an increasing viscosity, showing that the dynamics of the trehalose-water matrix and the stability of the native structure of the protein are not necessarily coupled. We also infer, by analyzing the maximum amount of water for which ice formation is avoided, that the preferential hydration model is consistent with our experimental data.

Slow Debye-type peak observed in the dielectric response of polyalcohols

Dielectric relaxation spectroscopy of glass forming liquids normally exhibits a relaxation scenario that seems to be surprisingly general. However, the relaxation dynamics is more complicated for hydrogen bonded liquids. For instance, the dielectric response of monoalcohols is dominated by a mysterious Debye-like process at lower frequencies than the structural alpha-relaxation that is normally dominating the spectra of glass formers. For polyalcohols this process has been thought to be absent or possibly obscured by a strong contribution from conductivity and polarization effects at low frequencies. We here show that the Debye-like process, although much less prominent, is also present in the response of polyalcohols. It can be observed in the derivative of the real part of the susceptibility or directly in the imaginary part if the conductivity contribution is reduced by covering the upper electrode with a thin Teflon layer. We report on results from broadband dielectric spectroscopy studies of several polyalcohols: glycerol, xylitol, and sorbitol. The findings are discussed in relation to other experimental observations of ultraslow (i.e., slower than the viscosity related alpha-relaxation) dynamics in glass formers.

Different behavior of water in confined solutions of high and low solute concentrations

Water-glycerol solutions confined in 21 Å pores of the silica matrix MCM-41 C10 have been studied using differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). The results suggest a micro-phase separation caused by the confinement. Likely the water molecules coordinate to the hydroxyl surface groups of the pores, leaving most of the glycerol molecules in the centre of the pores. This makes the dynamics of glycerol almost concentration independent up to water concentrations of about 85 wt%. However, at higher water concentrations no substantial clustering of glycerol molecules should occur and the glass transition related dynamics exhibit an anomalous behaviour. Instead of a common plasticization effect of water, as for the corresponding bulk solutions (when no ice is formed), it is evident that water acts as an anti-plasticizer in the confinement at high water concentrations. We propose that the increased water concentration slows down the glass transition related dynamics in the deeply supercooled regime due to that a rigid hydrogen bonded network structure of water molecules is formed at low temperatures and low glycerol concentrations. This is in contrast to the situation in a homogenously mixed bulk solution of a high solute concentration where the water molecules will be less hydrogen bonded, and therefore are typically more mobile than the surrounding solute molecules. An almost complete hydrogen bonded network of water molecules may, even in confinements, be sufficiently rigid to slow down the relaxation of embedded solute molecules. It can also be expressed the other way around, i.e. small amounts of glycerol act as a plasticizer for water, due to its breaking up of the nearly tetrahedral network structure. From the here observed concentration dependent behaviour of the deeply supercooled bulk and confined solutions it seems, furthermore, evident that the Tg value of bulk water cannot be estimated from extrapolations of aqueous solutions.

Calorimetric and relaxation properties of xylitol-water mixtures

We present the first broadband dielectric spectroscopy (BDS) and differential scanning calorimetry study of supercooled xylitol-water mixtures in the whole concentration range and in wide frequency (10(-2)-10(6) Hz) and temperature (120-365 K) ranges. The calorimetric glass transition, T(g), decreases from 247 K for pure xylitol to about 181 K at a water concentration of approximately 37 wt. %. At water concentrations in the range 29-35 wt. % a plentiful calorimetric behaviour is observed. In addition to the glass transition, almost simultaneous crystallization and melting events occurring around 230-240 K. At higher water concentrations ice is formed during cooling and the glass transition temperature increases to a steady value of about 200 K for all higher water concentrations. This T(g) corresponds to an unfrozen xylitol-water solution containing 20 wt. % water. In addition to the true glass transition we also observed a glass transition-like feature at 220 K for all the ice containing samples. However, this feature is more likely due to ice dissolution [A. Inaba and O. Andersson, Thermochim. Acta, 461, 44 (2007)]. In the case of the BDS measurements the presence of water clearly has an effect on both the cooperative α-relaxation and the secondary β-relaxation. The α-relaxation shows a non-Arrhenius temperature dependence and becomes faster with increasing concentration of water. The fragility of the solutions, determined by the temperature dependence of the α-relaxation close to the dynamic glass transition, decreases with increasing water content up to about 26 wt. % water, where ice starts to form. This decrease in fragility with increasing water content is most likely caused by the increasing density of hydrogen bonds, forming a network-like structure in the deeply supercooled regime. The intensity of the secondary β-relaxation of xylitol decreases noticeably already at a water content of 2 wt. %, and at a water content above 5 wt. % it has been replaced by a considerably stronger water (w) relaxation at about the same frequency. However, the similarities in time scale and activation energy between the w-relaxation and the β-relaxation of xylitol at water contents below 13 wt. % suggest that the w-relaxation is governed, in some way, by the β-relaxation of xylitol, since clusters of water molecules are rare at these water concentrations. At higher water concentrations the intensity and relaxation rate of the w-relaxation increase rapidly with increasing water content (up to the concentration where ice starts to form), most likely due to a rapid increase of small water clusters where an increasing number of water molecules interacting with other water molecules.

Cause of the fragile-to-strong transition observed in water confined in C-S-H gel

In this study, the rotational dynamics of hydration water confined in calcium-silicate-hydrate (C-S-H) gel with a water content of 22 wt.% was studied by broadband dielectric spectroscopy in broad temperature (110-300 K) and frequency (10(-1)-10(8) Hz) ranges. The C-S-H gel was used as a 3D confining system for investigating the possible existence of a fragile-to-strong transition for water around 220 K. Such transition was observed at 220 K in a previous study [Y. Zhang, M. Lagi, F. Ridi, E. Fratini, P. Baglioni, E. Mamontov and S. H. Chen, J. Phys.: Condens. Matter 20, 502101 (2008)] on a similar system, and it was there associated with a hidden critical point of bulk water. However, based on the experimental results presented here, there is no sign of a fragile-to-strong transition for water confined in C-S-H gel. Instead, the fragile-to-strong transition can be explained by a merging of two different relaxation processes at about 220 K.