Effects of the Low-Temperature Transitions of Confined Water on the Structures of Isolated and Cytoplasmic Proteins (original) (raw)
Related papers
Proteins in frozen solutions: evidence of ice-induced partial unfolding
Biophysical Journal, 1996
From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphorescence properties nor the catalytic activity. The variation in the lifetime parameter was found to be a smooth function of the residual volume of liquid water in equilibrium with ice and to depend on the morphology of ice. The addition of cryoprotectants such as glycerol and sucrose profoundly attenuates or even eliminates the perturbation. These results are interpreted in terms of adsorption of protein molecules onto the surface of ice. . Abbreviations used: T, Trp phosphorescence lifetime; LADH, alcohol dehydrogenase from horse liver; AP, alkaline phosphatase from E. coli; GAPDH, glyceraldehyde-3-phosphate dehydrogenase from yeast; LDH, lactic dehydrogenase from rabbit muscle.
Microscopic Mechanism of Protein Cryopreservation in an Aqueous Solution with Trahalose
In order to investigate the cryoprotective mechanism of trehalose on proteins, we use molecular dynamics computer simulations to study the microscopic dynamics of water upon cooling in an aqueous solution of lysozyme and trehalose. We find that the presence of trehalose causes global retardation of the dynamics of water. Comparing aqueous solutions of lysozyme with/without trehalose, we observe that the dynamics of water in the hydration layers close to the protein is dramatically slower when trehalose is present in the system. We also analyze the structure of water and trehalose around the lysozyme and find that the trehalose molecules form a cage surrounding the protein that contains very slow water molecules. We conclude that the transient cage of trehalose molecules that entraps and slows the water molecules prevents the crystallisation of protein hydration water upon cooling.
Observation of ice-like water layers at an aqueous protein surface
Proceedings of the National Academy of Sciences, 2014
We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0°C to −2°C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice.
Biophysical Chemistry, 2009
The influence of proteins and solutes on hysteresis of freezing and melting of water was measured by infrared (IR) spectroscopy. Of the solutes examined, poly-L-arginine and flounder antifreeze protein produced the largest freezing point depression of water, with little effect on the melting temperature. Poly-L-lysine, poly-Lglutamate, cytochrome c and bovine serum albumin had less effect on the freezing of water. Small compounds used to mimic non-polar (trimethylamine N-oxide, methanol), positively charged (guanidinium chloride, NH 4 Cl, urea) and negatively charged (Na acetate) groups on protein surfaces were also examined. These molecules and ions depress water's freezing point and the melting profiles became broad. Since infrared absorption measures both bulk solvent and solvent bound to the solutes, this result is consistent with solutes interacting with liquid water. The amide I absorption bands of antifreeze protein and poly-Larginine do not detectably change with the phase transition of water. An interpretation is that the antifreeze protein and poly-L-arginine order liquid water such that the water around the group is ice-like.
Ice-induced partial unfolding and aggregation of an integral membrane protein
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253 K, with t ½ = 3.9 ± 0.6 h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca 2+ -ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2 ± 0.3 (fast cooling) and 3.7 ± 0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.
The Journal of Physical Chemistry B, 2011
Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous proteinÀsaccharideÀwater systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehaloseÀwater systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehaloseÀwater systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T g) and the functional properties of the encapsulated protein (thermal denaturation, T den). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehaloseÀwater solvent. Conversely, at low hydration, lower T g values are measured in the presence of proteins: the lack of water promotes sugarÀprotein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T den increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T g and T den is also observed, in line with previous results; this finding suggests that collective waterÀtrehalose interactions, responsible for the glass transition, also influence the protein denaturation.
Electron microscopy and calorimetry of proteins in supercooled water
Scientific Reports
Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice.
Water Thermodynamics and Its Effects on the Protein Stability and Activity
Biophysica, 2021
We discuss a phenomenon regarding water that was until recently a subject of scientific interest: i.e., the dynamical crossover, from the fragile to strong glass forming material, for both bulk and protein hydration water. Such crossover is characterized by a temperature TL in which significant dynamical changes like the decoupling (or the violation of the Stokes-Einstein relation) of homologous transport parameters, e.g., the density relaxation time τ and the viscosity η, occur in the system. On this respect we considered the dynamic properties of water-protein systems. More precisely, we focused our study on proteins and their hydration water, as far as bulk and confined water. In order to clarify the effects of the water dynamical crossover on the protein properties we considered and discussed in a comparative way previous and new experimental data, obtained from different techniques and molecular dynamic simulation (MD). We pointed out the reasons for different dynamical finding...
Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures
Acta Crystallographica Section D Biological Crystallography, 2001
The solvent behaviour of¯ash-cooled protein crystals was studied in the range 100±180 K by X-ray diffraction. If the solvent is within large channels it crystallizes at 155 K, as identi®ed by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar amount of solvent is con®ned to narrow channels and/or individual cavities it does not crystallize in the studied temperature range. It is concluded that the solvent in large channels behaves similarly to bulk water, whereas when con®ned to narrow channels it is mainly protein-associated. The analogy with the behaviour of pure bulk water provides circumstantial evidence that only solvent in large channels undergoes a glass transition in the 100±180 K temperature range. These studies reveal that¯ash-cooled protein crystals are arrested in a metastable state up to at least 155 K, thus providing an upper temperature limit for their storage and handling. The results are pertinent to the development of rational crystal annealing procedures and to the study of temperature-dependent radiation damage to proteins. Furthermore, they suggest an experimental paradigm for studying the correlation between solvent behaviour, protein dynamics and protein function.