| 1. |
- Davidovic, Monika, et al.
(författare)
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Protein cold denaturation as seen from the solvent
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:3, s. 1025-1036
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Tidskriftsartikel (refereegranskat)abstract
- Unlike most ordered molecular systems, globular proteins exhibit a temperature of maximum stability, implying that the structure can be disrupted by cooling. This cold denaturation phenomenon is usually linked to the temperature-dependent hydrophobic driving force for protein folding. Yet, despite the key role played by protein−water interactions, hydration changes during cold denaturation have not been investigated experimentally. Here, we use water−17O spin relaxation to monitor the hydration dynamics of the proteins BPTI, ubiquitin, apomyoglobin, and β-lactoglobulin in aqueous solution from room temperature down to −35 °C. To access this temperature range without ice formation, we contained the protein solution in nonperturbing picoliter emulsion droplets. Among the four proteins, only the destabilized apomyoglobin was observed to cold denature. Ubiquitin was found to be thermodynamically stable at least down to −32 °C, whereas β-lactoglobulin is expected to be unstable below −5 °C but remains kinetically trapped in the native state. When destabilized by 4 M urea, β-lactoglobulin cold denatures at 10 °C, as found previously by other methods. As seen from the solvent, the cold-denatured states of apomyoglobin in water and β-lactoglobulin in 4 M urea are relatively compact and are better described as solvent-penetrated than as unfolded. This finding challenges the popular analogy between cold denaturation and the anomalous low-temperature increase in aqueous solubility of nonpolar molecules. Our results also suggest that the reported cold denaturation at −20 °C of ubiquitin encapsulated in reverse micelles is caused by the low water content rather than by the low temperature.
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| 2. |
- Kadhirvel, Saraboji, et al.
(författare)
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The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 296-306
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Tidskriftsartikel (refereegranskat)abstract
- The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.
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| 3. |
- Mattea, Carlos, et al.
(författare)
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Dynamics at the protein-water interface from 17O spin relaxation in deeply supercooled solutions
- 2008
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 95:6, s. 2951-2963
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Tidskriftsartikel (refereegranskat)abstract
- Most of the decisive molecular events in biology take place at the protein-water interface. The dynamical properties of the hydration layer are therefore of fundamental importance. To characterize the dynamical heterogeneity and rotational activation energy in the hydration layer, we measured the 17O spin relaxation rate in dilute solutions of three proteins in a wide temperature range extending down to 238 K. We find that the rotational correlation time can be described by a power-law distribution with exponent 2.1 – 2.3. Except for a small fraction of secluded hydration sites, the dynamic perturbation in the hydration layer is the same for all proteins and does not differ in any essential way from the hydration shell of small organic solutes. In both cases, the dynamic perturbation factor is less than 2 at room temperature and exhibits a maximum near 260 K. This maximum implies that, at low temperatures, the rate of water molecule rotation has a weaker temperature dependence in the hydration layer than in bulk water. We attribute this difference to the temperature-independent constraints that the protein surface imposes on the water H-bond network. The free hydration layer studied here differs qualitatively from confined water in solid protein powder samples.
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| 4. |
- Modig, Kristofer, et al.
(författare)
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High water mobility on the ice-binding surface of a hyperactive antifreeze protein.
- 2010
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Ingår i: Physical chemistry chemical physics : PCCP. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 12:Online 29th July 2010, s. 10189-10197
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Tidskriftsartikel (refereegranskat)abstract
- Antifreeze proteins (AFPs) prevent uncontrolled ice formation in organisms exposed to subzero temperatures by binding irreversibly to specific planes of nascent ice crystals. To understand the thermodynamic driving forces and kinetic mechanism of AFP activity, it is necessary to characterize the hydration behavior of these proteins in solution. With this aim, we have studied the hyperactive insect AFP from Tenebrio molitor (TmAFP) with the (17)O magnetic relaxation dispersion (MRD) method, which selectively monitors the rotational motion and exchange kinetics of water molecules on picosecond-microsecond time scales. The global hydration behavior of TmAFP is found to be similar to non-antifreeze proteins, with no evidence of ice-like or long-ranged modifications of the solvent. However, two sets of structural water molecules, located within the core and on the ice-binding face in the crystal structure of TmAFP, may have functional significance. We find that 2 of the 5 internal water molecules exchange with a residence time of 8 +/- 1 mus at 300 K and a large activation energy of approximately 50 kJ mol(-1), reflecting intermittent large-scale conformational fluctuations in this exceptionally dense and rigid protein. Six water molecules arrayed with ice-like spacing in the central trough on the ice-binding face exchange with bulk water on a sub-nanosecond time scale. The combination of high order and fast exchange may allow these water molecules to contribute entropically to the ice-binding affinity without limiting the absorption rate.
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| 5. |
- Qvist, Johan, et al.
(författare)
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A dry ligand-binding cavity in a solvated protein
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:17, s. 6296-6301
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Tidskriftsartikel (refereegranskat)abstract
- Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine beta-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water (17)O and (2)H magnetic relaxation dispersion (MRD), (13)C NMR spectroscopy, molecular dynamics simulations, and free energy calculations to establish the absence of water from the binding cavity. Whereas carbon nanotubes of the same diameter are filled by a hydrogen-bonded water chain, the MRD data show that the binding pore in the apo protein is either empty or contains water molecules with subnanosecond residence times. However, the latter possibility is ruled out by the computed hydration free energies, so we conclude that the 315 A(3) binding pore is completely empty. The apo protein is thus poised for efficient binding of fatty acids and other nonpolar ligands. The qualitatively different hydration of the beta-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water-wall interactions and water entropy.
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| 6. |
- Qvist, Johan, et al.
(författare)
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Hydration Dynamics of a Halophilic Protein in Folded and Unfolded States
- 2012
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 116:10, s. 3436-3444
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Tidskriftsartikel (refereegranskat)abstract
- Proteins from halophilic microorganisms thriving at high salinity have an excess of charged carboxylate groups, and it is widely believed that this gives rise to an exceptionally strong hydration that stabilizes these proteins against unfolding and aggregation. Here, we examine this hypothesis by characterizing the hydration dynamics of a halophilic model protein with frequency- and temperature-dependent O-17 magnetic relaxation. The halophilic protein Kx6E was constructed by replacing six lysine residues with glutamate residues in the IgG binding domain of protein L. We also studied the unfolded form of Kx6E in the absence of salt. We find that the hydration dynamics of Kx6E does not differ from protein L or from other previously studied mesophilic proteins. This finding challenges the hypothesis of exceptional hydration for halophilic proteins. The unfolded form of Kx6E is found to be expanded, with a weaker dynamical perturbation of the hydration water than for folded proteins.
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| 7. |
- Qvist, Johan, et al.
(författare)
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Rotational dynamics in supercooled water from nuclear spin relaxation and molecular simulations.
- 2012
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 136:20
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Tidskriftsartikel (refereegranskat)abstract
- Structural dynamics in liquid water slow down dramatically in the supercooled regime. To shed further light on the origin of this super-Arrhenius temperature dependence, we report high-precision (17)O and (2)H NMR relaxation data for H(2)O and D(2)O, respectively, down to 37 K below the equilibrium freezing point. With the aid of molecular dynamics (MD) simulations, we provide a detailed analysis of the rotational motions probed by the NMR experiments. The NMR-derived rotational correlation time τ(R) is the integral of a time correlation function (TCF) that, after a subpicosecond librational decay, can be described as a sum of two exponentials. Using a coarse-graining algorithm to map the MD trajectory on a continuous-time random walk (CTRW) in angular space, we show that the slowest TCF component can be attributed to large-angle molecular jumps. The mean jump angle is ∼48° at all temperatures and the waiting time distribution is non-exponential, implying dynamical heterogeneity. We have previously used an analogous CTRW model to analyze quasielastic neutron scattering data from supercooled water. Although the translational and rotational waiting times are of similar magnitude, most translational jumps are not synchronized with a rotational jump of the same molecule. The rotational waiting time has a stronger temperature dependence than the translation one, consistent with the strong increase of the experimentally derived product τ(R) D(T) at low temperatures. The present CTRW jump model is related to, but differs in essential ways from the extended jump model proposed by Laage and co-workers. Our analysis traces the super-Arrhenius temperature dependence of τ(R) to the rotational waiting time. We present arguments against interpreting this temperature dependence in terms of mode-coupling theory or in terms of mixture models of water structure.
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| 8. |
- Qvist, Johan, et al.
(författare)
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Structural dynamics of supercooled water from quasielastic neutron scattering and molecular simulations.
- 2011
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 134:14
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Tidskriftsartikel (refereegranskat)abstract
- One of the outstanding challenges presented by liquid water is to understand how molecules can move on a picosecond time scale despite being incorporated in a three-dimensional network of relatively strong H-bonds. This challenge is exacerbated in the supercooled state, where the dramatic slowing down of structural dynamics is reminiscent of the, equally poorly understood, generic behavior of liquids near the glass transition temperature. By probing single-molecule dynamics on a wide range of time and length scales, quasielastic neutron scattering (QENS) can potentially reveal the mechanistic details of water's structural dynamics, but because of interpretational ambiguities this potential has not been fully realized. To resolve these issues, we present here an extensive set of high-quality QENS data from water in the range 253-293 K and a corresponding set of molecular dynamics (MD) simulations to facilitate and validate the interpretation. Using a model-free approach, we analyze the QENS data in terms of two motional components. Based on the dynamical clustering observed in MD trajectories, we identify these components with two distinct types of structural dynamics: picosecond local (L) structural fluctuations within dynamical basins and slower interbasin jumps (J). The Q-dependence of the dominant QENS component, associated with J dynamics, can be quantitatively rationalized with a continuous-time random walk (CTRW) model with an apparent jump length that depends on low-order moments of the jump length and waiting time distributions. Using a simple coarse-graining algorithm to quantitatively identify dynamical basins, we map the Newtonian MD trajectory on a CTRW trajectory, from which the jump length and waiting time distributions are computed. The jump length distribution is Gaussian and the rms jump length increases from 1.5 to 1.9 Å as the temperature increases from 253 to 293 K. The rms basin radius increases from 0.71 to 0.75 Å over the same range. The waiting time distribution is exponential at all investigated temperatures, ruling out significant dynamical heterogeneity. However, a simulation at 238 K reveals a small but significant dynamical heterogeneity. The macroscopic diffusion coefficient deduced from the QENS data agrees quantitatively with NMR and tracer results. We compare our QENS analysis with existing approaches, arguing that the apparent dynamical heterogeneity implied by stretched exponential fitting functions results from the failure to distinguish intrabasin (L) from interbasin (J) structural dynamics. We propose that the apparent dynamical singularity at ∼220 K corresponds to freezing out of J dynamics, while the calorimetric glass transition corresponds to freezing out of L dynamics.
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| 9. |
- Qvist, Johan, et al.
(författare)
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Thermal signature of hydrophobic hydration dynamics
- 2008
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 130:31, s. 10345-10353
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic hydration, the perturbation of the aqueous solvent near an apolar solute or interface, is a fundamental ingredient in many chemical and biological processes. Both bulk water and aqueous solutions of apolar solutes behave anomalously at low temperatures for reasons that are not fully understood. Here, we use 2H NMR relaxation to characterize the rotational dynamics in hydrophobic hydration shells over a wide temperature range, extending down to 243 K. We examine four partly hydrophobic solutes: the peptides N-acetyl-glycine-N′-methylamide and N-acetyl-leucine-N′-methylamide, and the osmolytes trimethylamine N-oxide and tetramethylurea. For all four solutes, we find that water rotates with lower activation energy in the hydration shell than in bulk water below 255 2 K. At still lower temperatures, water rotation is predicted to be faster in the shell than in bulk. We rationalize this behavior in terms of the geometric constraints imposed by the solute. These findings reverse the classical “iceberg” view of hydrophobic hydration by indicating that hydrophobic hydration water is less ice-like than bulk water. Our results also challenge the “structural temperature” concept. The two investigated osmolytes have opposite effects on protein stability but have virtually the same effect on water dynamics, suggesting that they do not act indirectly via solvent perturbations. The NMR-derived picture of hydrophobic hydration dynamics differs substantially from views emerging from recent quasielastic neutron scattering and pump−probe infrared spectroscopy studies of the same solutes. We discuss the possible reasons for these discrepancies.
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| 10. |
- Qvist, Johan, et al.
(författare)
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Time scales of water dynamics at biological interfaces: peptides, proteins and cells
- 2009
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Ingår i: Faraday Discussions. - : Royal Society of Chemistry (RSC). - 1364-5498. ; 141:1, s. 131-144
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Tidskriftsartikel (refereegranskat)abstract
- Water 2H and 17O spin relaxation is used to study water dynamics in the hydration layers of two small peptides, two globular proteins and in living cells of two microorganisms. The dynamical heterogeneity of hydration water is characterized by performing relaxation measurements over a wide temperature range, extending deeply into the supercooled regime, or by covering a wide frequency range. Protein hydration layers can be described by a power-law distribution of rotational correlation times with an exponent close to 2. This distribution comprises a small fraction of protein-specific hydration sites, where water rotation is strongly retarded, and a dominant fraction of generic hydration sites, where water rotation is as fast as in the hydration shells of small peptides. The generic dynamic perturbation factor is less than 2 at room temperature and exhibits a maximum near 260 K. The dynamic perturbation is induced by H-bond constraints that interfere with the cooperative mechanism that facilitates rotation in bulk water. Because these constraints are temperature-independent, hydration water does not follow the super-Arrhenius temperature dependence of bulk water. Water in living cells behaves as expected from studies of simpler model systems, the only difference being a larger fraction of secluded (strongly perturbed) hydration sites associated with the supramolecular organization in the cell. Intracellular water that is not in direct contact with biopolymers has essentially the same dynamics as bulk water. There is no significant difference in cell water dynamics between mesophilic and halophilic organisms, despite the high K+ and Na+ concentrations in the latter.
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