[Show abstract][Hide abstract] ABSTRACT: Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.
Proceedings of the National Academy of Sciences 07/2015; 112(33). DOI:10.1073/pnas.1506079112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipid-binding proteins sequester amphiphilic molecules in a large internal cavity occupied by ∼ 30 water molecules, some of which are displaced by the ligand. The role of these internal water molecules in lipid binding and release is not understood. We use magnetic relaxation dispersion (MRD) to directly monitor internal-water dynamics in apo and palmitate-bound rat intestinal fatty acid-binding protein (rIFABP). Specifically, we record the water 2H and 17O MRD profiles of the apo and holo forms of rIFABP in solution or immobilized by covalent cross-links. A global analysis of this extensive data set identifies three internal-water classes with mean survival times of ∼ 1 ns, ∼ 100 ns and ∼ 6 μs. We associate the two longer time scales with conformational fluctuations of the gap between β-strands D and E (∼ 6 μs) and of the portal at the helix-capped end of the β-barrel (∼ 100 ns). These fluctuations limit the exchange rates of a few highly ordered structural water molecules, but not the dissociation rate of the fatty acid. The remaining 90% (apo) or 70% (holo) of cavity waters exchange among internal hydration sites on a time scale of ∼ 1 ns but exhibit substantial orientational order, particularly in the holo form.
The Journal of Physical Chemistry B 05/2015; 119(25). DOI:10.1021/acs.jpcb.5b03214 · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigate protein-protein interactions in solution by small-angle X-ray scattering (SAXS) and theoretical modeling. The structure factor for solutions of bovine pancreatic trypsin inhibitor (BPTI), myoglobin (Mb), and intestinal fatty acid-binding protein (IFABP) is determined from SAXS measurements at multiple concentrations, from Monte Carlo simulations with a coarse-grained structure-based interaction model, and from analytic approximate solutions of two idealized colloidal interaction models without adjustable parameters. By combining these approaches, we find that the structure factor is essentially determined by hard-core and screened electrostatic interactions. Other soft short-ranged interactions (van der Waals and solvation-related) are either individually insignificant or tend to cancel out. The structure factor is also not significantly affected by charge fluctuations. For Mb and IFABP, with a small net charge and relatively symmetric charge distribution, the structure factor is well described by a hard-sphere model. For BPTI, with a larger net charge, screened electrostatic repulsion is also important, but the asymmetry of the charge distribution reduces the repulsion from that predicted by a charged hard-sphere model with the same net charge. Such charge asymmetry may also amplify the effect of shape asymmetry on the protein-protein potential of mean force.
The Journal of Physical Chemistry B 08/2014; 118(34):10111-19. DOI:10.1021/jp505809v · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glutaraldehyde (GA) reacts with amino groups in proteins, forming intermolecular cross-links that, at sufficiently high protein concentration, can transform a protein solution into a gel. Although GA has been used as a cross-linking reagent for decades, neither the cross-linking chemistry nor the microstructure of the resulting protein gel have been clearly established. Here we use small-angle X-ray scattering (SAXS) to characterise the microstructure and structural kinetics of gels formed by cross-linking of pancreatic trypsin inhibitor, myoglobin or intestinal fatty acid-binding protein. By comparing the scattering from gels and dilute solutions, we extract the structure factor and the pair correlation function of the gels. The protein gels are spatially heterogeneous, with dense clusters linked by sparse networks. Within the clusters, adjacent protein molecules are almost in contact, but the protein concentration in the cluster is much lower than in a crystal. At the ∼1 nm SAXS resolution, the native protein structure is unaffected by cross-linking. The cluster radius is in the range 10-50 nm, with the cluster size determined mainly by the availability of lysine amino groups on the protein surface. The development of structure in the gel, on time scales from minutes to hours, appears to obey first-order kinetics. Cross-linking is slower at acidic pH, where the population of amino groups in the reactive deprotonated form is low. These results support the use of cross-linked protein gels in NMR studies of protein dynamics and for modeling NMR relaxation in biological tissue.
[Show abstract][Hide abstract] ABSTRACT: Myoglobin (Mb) binds diatomic ligands, like O2, CO, and NO, in a cavity that is only transiently accessible. Crystallography and molecular simulations show that the ligands can migrate through an extensive network of transiently connected cavities, but disagree on the locations and occupancy of internal hydration sites. Here, we use water (2)H and (17)O magnetic relaxation dispersion (MRD) to characterize the internal water molecules in Mb under physiological conditions. We find that equine carbonmonoxy Mb contains 4.5 ± 1.0 ordered internal water molecules with a mean survival time of 5.6 ± 0.5 μs at 25 °C. The likely location of these water molecules are the four polar hydration sites, including one of the xenon-binding cavities, that are fully occupied in all high-resolution crystal structures of equine Mb. The finding that water escapes from these sites, located 17 - 31 Å apart in the protein, on the same μs time scale suggests a global exchange mechanism. We propose that this mechanism involves transient penetration of the protein by H-bonded water chains. Such a mechanism could play a functional role by eliminating trapped ligands. In addition, the MRD results indicate that two or three of the 11 histidine residues of equine Mb undergo intramolecular hydrogen exchange on a μs time scale.
The Journal of Physical Chemistry B 11/2013; 117(47):14676-87. DOI:10.1021/jp409234g · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial spores in a metabolically dormant state can survive long periods without nutrients under extreme environmental conditions. The molecular basis of spore dormancy is not well understood, but the distribution and physical state of water within the spore is thought to play an important role. Two scenarios have been proposed for the spore's core region, containing the DNA and most enzymes. In the gel scenario, the core is a structured macromolecular framework permeated by mobile water. In the glass scenario, the entire core, including the water, is an amorphous solid and the quenched molecular diffusion accounts for the spore's dormancy and thermal stability. Here, we use (2)H magnetic relaxation dispersion to selectively monitor water mobility in the core of Bacillus subtilis spores in the presence and absence of core Mn(2+) ions. We also report and analyze the solid-state (2)H NMR spectrum from these spores. Our NMR data clearly support the gel scenario with highly mobile core water (∼25 ps average rotational correlation time). Furthermore, we find that the large depot of manganese in the core is nearly anhydrous, with merely 1.7% on average of the maximum sixfold water coordination.
[Show abstract][Hide abstract] ABSTRACT: In complex biological or colloidal samples, magnetic relaxation dispersion (MRD) experiments using the field-cycling technique can characterize molecular motions on time scales ranging from nanoseconds to microseconds, provided that a rigorous theory of nuclear spin relaxation is available. In gels, cross-linked proteins, and biological tissues, where an immobilized macromolecular component coexists with a mobile solvent phase, nuclear spins residing in solvent (or cosolvent) species relax predominantly via exchange-mediated orientational randomization (EMOR) of anisotropic nuclear (electric quadrupole or magnetic dipole) couplings. The physical or chemical exchange processes that dominate the MRD typically occur on a time scale of microseconds or longer, where the conventional perturbation theory of spin relaxation breaks down. There is thus a need for a more general relaxation theory. Such a theory, based on the stochastic Liouville equation (SLE) for the EMOR mechanism, is available for a single quadrupolar spin I = 1. Here, we present the corresponding theory for a dipole-coupled spin-1/2 pair. To our knowledge, this is the first treatment of dipolar MRD outside the motional-narrowing regime. Based on an analytical solution of the spatial part of the SLE, we show how the integral longitudinal relaxation rate can be computed efficiently. Both like and unlike spins, with selective or non-selective excitation, are treated. For the experimentally important dilute regime, where only a small fraction of the spin pairs are immobilized, we obtain simple analytical expressions for the auto-relaxation and cross-relaxation rates which generalize the well-known Solomon equations. These generalized results will be useful in biophysical studies, e.g., of intermittent protein dynamics. In addition, they represent a first step towards a rigorous theory of water (1)H relaxation in biological tissues, which is a prerequisite for unravelling the molecular basis of soft-tissue contrast in clinical magnetic resonance imaging.
The Journal of Chemical Physics 10/2013; 139(14):144203. DOI:10.1063/1.4824105 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MD simulations can now explore the complex dynamics of proteins and their associated solvent in atomic detail on a millisecond time scale. Among the phenomena that thereby become amenable to detailed study are intermittent conformational transitions where the protein accesses transient high-energy states that often play key roles in biology. Here, we present a coherent theoretical framework, based on the stochastic theory of stationary point processes, that allows the essential dynamical characteristics of such processes to be efficiently extracted from the MD trajectory without assuming Poisson statistics. Since the complete information content of a point process is contained in the sequence of residence or interevent times, the experimentally relevant survival correlation function can be computed several orders of magnitude more efficiently than with the conventional approach, involving averaging over initial times. We also present a detailed analysis of the statistical and binning errors, of particular importance when MD results are compared with experiment. As an illustration of the general theoretical framework, we use a 1 ms MD trajectory of the protein BPTI to analyze the exchange kinetics of an internal water molecule and the dynamics of the rare conformational fluctuations that govern the rate of this exchange process.
Journal of Chemical Theory and Computation 05/2013; 9(6):2838–2848. DOI:10.1021/ct400161u · 5.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many proteins rely for their function on rare structural fluctuations whereby solvent and other small molecules gain transient access to internal cavities. In magnetic relaxation dispersion (MRD) experiments, water molecules buried in such cavities are used as intrinsic probes of the intermittent protein motions that govern their exchange with external solvent. While this has allowed a detailed characterization of exchange kinetics for several proteins, little is known about the exchange mechanism. Here, we use a millisecond all-atom MD trajectory produced by Shaw et al. (Science 2010, 330, 341) to characterize water exchange from the 4 internal hydration sites in the protein BPTI. Using a recently developed stochastic point process approach, we compute the survival correlation function probed by MRD experiments as well as other quantities designed to validate the exchange-mediated orientational randomization (EMOR) model used to interpret the MRD data. The EMOR model is found to be quantitatively accurate and the simulation reproduces the experimental mean survival times for all 4 sites with activation energy discrepancies in the range 0 - 3 kBT. On the other hand, the simulated hydration sites are somewhat too flexible and the water flip barrier is underestimated by up to 6 kBT. The simulation reveals that water molecules gain access to the internal sites by a transient aqueduct mechanism, migrating as single-file water chains through transient (< 5 ns) tunnels or pores. The present study illustrates the power of state-of-the-art MD simulations in validating and extending experimental results.
Journal of the American Chemical Society 05/2013; 106(2). DOI:10.1021/ja403405d · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The disaccharide trehalose stabilizes proteins against unfolding, but the underlying mechanism is not well understood. Because trehalose is preferentially excluded from the protein surface, it is of interest to examine how trehalose modifies the structure and dynamics of the solvent. From the spin relaxation rates of deuterated trehalose and (17)O-enriched water, we obtain the rotational dynamics of trehalose and water in solutions over wide ranges of concentration (0.025-1.5 M) and temperature (236-293 K). The results reveal direct solute-solute interactions at all concentrations, consistent with transient trehalose clusters. Similar to other organic solutes, the trehalose perturbation of water rotation (and hydrogen-bond exchange) is modest: a factor 1.6 (at 298 K) on average for the 47 water molecules in the first hydration layer. The deviation of the solute tumbling time from the Stokes-Einstein-Debye relation is partly caused by a dynamic solvent effect that is often modeled by incorporating "bound water" in the hydrodynamic volume. By comparing the measured temperature dependences of trehalose and water dynamics, we demonstrate that a more realistic local viscosity model accounts for this second-order dynamic coupling.
The Journal of Physical Chemistry B 07/2012; 116(30):9196-207. DOI:10.1021/jp304982c · 3.30 Impact Factor
[Show abstract][Hide abstract] 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.
The Journal of Chemical Physics 05/2012; 136(20):204505. DOI:10.1063/1.4720941 · 2.95 Impact Factor
[Show abstract][Hide abstract] 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 (17)O 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.
The Journal of Physical Chemistry B 03/2012; 116(10):3436-44. DOI:10.1021/jp3000569 · 3.30 Impact Factor
[Show abstract][Hide abstract] 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.
The Journal of Chemical Physics 04/2011; 134(14):144508. DOI:10.1063/1.3578472 · 2.95 Impact Factor
[Show abstract][Hide abstract] 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 micros 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.
[Show abstract][Hide abstract] ABSTRACT: A resonant enhancement of the water-1H relaxation rate at three distinct frequencies in the range 0.5-3 MHz has been observed in a variety of aqueous biological systems. These so-called quadrupole (Q) peaks have been linked to a dipolar flip-flop polarization transfer from 1H nuclei to rapidly relaxing amide 14N nuclei in rotationally immobilized proteins. While the Q-peak frequencies conform to the known amide 14N quadrupole coupling parameters, a molecular model that accounts for the intensity and shape of the Q peaks has not been available. Here, we present such a model and test it against an extensive set of Q-peak data from two fully hydrated crosslinked proteins under conditions of variable temperature, pH and H/D isotope composition. We propose that polarization transfer from bulk water to amide 14N occurs in three steps: from bulk water to a so-called intermediary proton via material diffusion/exchange, from intermediary to amide proton by cross-relaxation driven by exchange-mediated orientational randomization of their mutual dipole coupling, and from amide proton to 14N by resonant dipolar relaxation 'of the second kind', driven by 14N spin fluctuations, which, in turn, are induced by restricted rigid-body motions of the protein. An essentially equivalent description of the last step can be formulated in terms of coherent 1H-->14N polarization transfer followed by fast 14N relaxation. Using independent structural and kinetic information, we show that the Q peaks from these two proteins involve approximately 7 intermediary protons in internal water molecules and side-chain hydroxyl groups with residence times of order 10(-5) s. The model not only accounts quantitatively for the extensive data set, but also explains why Q peaks are hardly observed from gelatin gels.
Journal of Magnetic Resonance 04/2010; 203(2):257-73. DOI:10.1016/j.jmr.2010.01.008 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NMR relaxation experiments have provided a wealth of information about molecular motions in macromolecules and ordered fluids. Even though a rigorous theory of spin relaxation is available, the complexity of the investigated systems often makes the interpretation of limited datasets challenging and ambiguous. To allow physically meaningful information to be extracted from the data without commitment to detailed dynamical models, several versions of a model-free (MF) approach to data analysis have been developed. During the past 2 decades, the MF approach has been used in the vast majority of all NMR relaxation studies of internal motions in proteins and other macromolecules, and it has also played an important role in studies of colloidal systems. Although the MF approach has been almost universally adopted, substantial disagreement remains about its physical foundations and range of validity. It is our aim here to clarify these issues. To this end, we first present rigorous derivations of the three well-known MF formulas for the time correlation function relevant for isotropic solutions. These derivations are more general than the original ones, thereby substantially extending the range of validity of the MF approach. We point out several common misconceptions and explain the physical significance of the approximations involved. In particular, we discuss symmetry requirements and the dynamical decoupling approximation that plays a key role in the MF approach. We also derive a new MF formula, applicable to anisotropic fluids and solids, including microcrystalline protein samples. The so-called slowly relaxing local structure (SRLS) model has been advanced as an alternative to the MF approach that does not require dynamical decoupling of internal and global motions. To resolve the existing controversy about the relative merits of the SRLS model and the MF approach, we formulate and solve a planar version of the SRLS model. The analytical solution of this model reveals the unphysical consequences of the symmetrical two-body Smoluchowski equation as applied to protein dynamics, thus refuting the widely held belief that the SRLS model is more accurate than the MF approach. The different results obtained by analyzing data with these two approaches therefore do not indicate the importance of dynamical coupling between internal and global motions. Finally, we explore the two principal mechanisms of dynamical coupling in proteins: torque-mediated and friction-mediated coupling. We argue by way of specific analytically solvable models that torque-mediated coupling (which the SRLS model attempts to capture) is unimportant because the relatively slow internal motions that might couple to the global motion tend to be intermittent (jumplike) in character, whereas friction-mediated coupling (which neither the SRLS model nor the MF approach incorporates) may be important for proteins with unstructured parts or flexibly connected domains.
The Journal of Chemical Physics 12/2009; 131(22):224507. DOI:10.1063/1.3269991 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To probe internal motions in proteins on the 10(-8)-10(-5) s time scale by NMR relaxation, it is necessary to eliminate protein tumbling. Here, we examine to what extent magnetic relaxation dispersion (MRD) experiments on the water (1)H resonance report on protein motions in this time window. We also perform a critical test of two physically distinct mechanisms that have been proposed to explain and interpret (1)H MRD profiles from immobilized proteins: the exchange-mediated orientational randomization (EMOR) mechanism and the two-phase spin-fracton (2PSF) mechanism. For these purposes, we report the (1)H MRD profiles from protonated and partially deuterated ubiquitin, cross-linked by glutaraldehyde. The EMOR approach, with the crystal structure of ubiquitin as input, accounts quantitatively for the MRD data and shows that hydroxyl-bearing side chains undergo large-amplitude motions on the microsecond time scale. In contrast, the 2PSF model, which attributes (1)H relaxation to small-amplitude backbone vibrations that propagate in a low-dimensional fractal space, fails qualitatively in describing the effect of H-->D substitution. These findings appear to resolve the long-standing controversy over the molecular basis of water-(1)H relaxation in systems containing rotationally immobilized macromolecules, including biological tissue.
Journal of the American Chemical Society 12/2009; 131(51):18214-5. DOI:10.1021/ja908144y · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bacterial spore, the hardiest known life form, can survive in a metabolically dormant state for many years and can withstand high temperatures, radiation, and toxic chemicals. The molecular basis of spore dormancy and resistance is not understood, but the physical state of water in the different spore compartments is thought to play a key role. To characterize this water in situ, we recorded the water (2)H and (17)O spin relaxation rates in D(2)O-exchanged Bacillus subtilis spores over a wide frequency range. The data indicate high water mobility throughout the spore, comparable with binary protein-water systems at similar hydration levels. Even in the dense core, the average water rotational correlation time is only 50 ps. Spore dormancy therefore cannot be explained by glass-like quenching of molecular diffusion but may be linked to dehydration-induced conformational changes in key enzymes. The data demonstrate that most spore proteins are rotationally immobilized, which may contribute to heat resistance by preventing heat-denatured proteins from aggregating irreversibly. We also find that the water permeability of the inner membrane is at least 2 orders of magnitude lower than for model membranes, consistent with the reported high degree of lipid immobilization in this membrane and with its proposed role in spore resistance to chemicals that damage DNA. The quantitative results reported here on water mobility and transport provide important clues about the mechanism of spore dormancy and resistance, with relevance to food preservation, disease prevention, and astrobiology.
Proceedings of the National Academy of Sciences 11/2009; 106(46):19334-9. DOI:10.1073/pnas.0908712106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The time-dependent fluorescence frequency shift of protein-attached probes has a much slower decay than that for the free probe. The decay times, ranging from 10 ps to several nanoseconds, have been attributed to hydration water motions several orders of magnitude slower than those in the hydration shell of small solutes. This interpretation deviates strongly from the prevailing picture of protein hydration dynamics. We argue here that the slow decay in the fluorescence shift can be explained by a ubiquitous solvent polarization mechanism, with no need to invoke slow water motions or a dynamic coupling with protein motions. This mechanism can be qualitatively understood with the aid of a dielectric continuum model. We therefore conclude that the long decay times measured with time-dependent fluorescence spectroscopy contain no information about protein hydration dynamics.
The Journal of Physical Chemistry B 07/2009; 113(24):8210-3. DOI:10.1021/jp9027589 · 3.30 Impact Factor