Femtochemistry of orange II in solution and in chemical and biological nanocavities

In this work, we report on studies of the nature of the dynamics and hydrophobic binding in cyclodextrins and human serum albumin protein complexes with orange II. With femtosecond time resolution, we examined the proton-transfer and trans-cis isomerization reactions of the ligand in these nanocavities and in pure solvents. Because of confinement at the ground state, the orientational motion in the formed phototautomer is restricted, leading to a rich dynamics. Therefore, the emission lifetimes span a large window of tens to hundreds of picoseconds in the cavities. Possible H-bond interactions between the guest and cyclodextrin do not affect the caged dynamics. For the protein-ligand complexes, slow diffusional motion ( approximately 630 ps) observed in the anisotropy decay indicates that the binding structure is not completely rigid, and the embedded guest is not frozen with the hydrophobic pocket. The ultrafast isomerization and decays are explained in terms of coupling motions between N-N and C-N stretching modes of the formed tautomer. We discuss the role of confinement on the trans-cis isomerization with the cavities and its relationships to frequency and time domains of nanostructure emission.     

Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.

Interactions between the erythrocyte membrane andits skeleton are mediated primarily by binding of cytoskeletal components to aconformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3).To examine the nanosecond segmental motions of cdb3, band 3 was labeledselectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3about which pH-dependent conformational changes occur. Time-resolved anisotropyof labeled cdb3 in isolated form and in stripped erythrocyte membranes wasmeasured by parallel-acquisition frequency-domain microfluorimetry. Samples hada single-component fluorescein lifetime of approximately 4 ns. Multifrequencyphase and modulation data (5-200 MHz) fitted well to a segmental motion modelcontaining two correlation times (tau 1c and tau 2c) and two limitinganisotropies (r1infinity and r2infinity). Measurements in protease-cleaved anddenatured samples indicated that tau 1c (100-150 ps) corresponded to rapidrotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmentalmotion of cdb3. Both motions were hindered as quantified by nonzero r1infinityand r2infinity. The strong pH dependence of segmental motion correlated withthat of cdb3 conformation measured by intrinsic tryptophan fluorescence.Significant changes in cdb3 segmental motion occurred upon interactions with thesmall ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymesknown to bind to the N terminus of band 3. These time-resolved fluorescencemeasurements of the nanosecond segmental dynamics of a labeled membrane proteinprovide evidence for the sensitivity of cdb3 conformation to ligand binding andsuggest long-range structural communication through cdb3.     

Global structural motions from the strain of a single hydrogen bond

The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the β-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120–H43–T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?     

Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gα proteins

Proteins with similar crystal structures can have dissimilar rates of substrate binding and catalysis. Here we used molecular dynamics simulations and biochemical analysis to determine the role of intradomain and interdomain motions in conferring distinct activation rates to two Gα proteins, Gα(i1) and GPA1. Despite high structural similarity, GPA1 can activate itself without a receptor, whereas Gα(i1) cannot. We found that motions in these proteins vary greatly in type and frequency. Whereas motion is greatest in the Ras domain of Gα(i1), it is greatest in helices αA and αB from the helical domain of GPA1. Using protein chimeras, we show that helix αA from GPA1 is sufficient to confer rapid activation to Gα(i1). Gα(i1) has less intradomain motion than GPA1 and instead displays interdomain displacement resembling that observed in a receptor-heterotrimer crystal complex. Thus, structurally similar proteins can have distinct atomic motions that confer distinct activation mechanisms.     

Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: the implication of molecular mechanisms of general anesthesia

Interactions of volatile anesthetics with the central nervous system are characterized by low yet specific binding affinities. Although neurotransmitter-gated ion channels are considered the primary anesthetic targets, the mechanism of action at the molecular level remains elusive. We consider here the theoretical implications of channel dynamics on anesthetic action in a simplified membrane-channel system. Large-scale 2.2-ns all-atom molecular dynamics simulations were performed to study the effects of halothane, a clinical anesthetic, on a gramicidin A (gA) channel in a fully hydrated dimyristoyl phosphatidylcholine membrane. In agreement with experimental results, anesthetics preferentially target the anchoring residues at the channel-lipid-water interface. Although the anesthetic effect on channel structure is minimal, the presence of halothane profoundly affects channel dynamics. For 2.2-ns simulation, the rms fluctuation of gA backbone in the lipid core increases from approximately equal 1 A in the absence of anesthetics to approximately equal 1.5 A in the presence of halothane. Autocorrelation analysis reveals that halothane (i) has no effect on the subpicosecond librational motion, (ii) prolongs the backbone autocorrelation time in the 10- to 100-ps time scale, and (iii) significantly decreases the asymptotic values of generalized order parameter and correlation time of nanosecond motions for the inner but not the outer residues. The simulation results discount the viewpoint of a structure-function paradigm that overrates the importance of structural fitting between general anesthetics and yet-unidentified hydrophobic protein pockets. Instead, the results underscore the global, as opposed to local, effects of anesthetics on protein dynamics as the underlying mechanisms for the action of general anesthetics and possibly of other low-affinity drugs.     

Heat capacity changes and hydrophobic interactions in the binding of FK506 and rapamycin to the FK506 binding protein.

Differential interactions among nonpolar moieties at protein/ligand interfaces, and of these nonpolar groups with water, collectively termed hydrophobic interactions, are widely believed to make important energetic contributions to the stability of protein/ligand complexes. Quantitative estimates of hydrophobic interactions, and an evaluation of their structural basis, are essential for obtaining structure-based predictions of the free energies of binding for the purpose of drug design. Two largely nonpolar, immunosuppressive agents, FK506 and rapamycin, each bind with high affinity to a common hydrophobic pocket on a small peptidylproline cis-trans isomerase known as FK506 binding protein (FKBP-12) and inhibit its activity. In an effort to elucidate the structural features of these ligands responsible for the observed energetics, we have undertaken an investigation of the thermodynamics of binding of FK506 and rapamycin to FKBP-12. Enthalpies of binding have been determined by high-precision titration calorimetry over a range of temperature, allowing estimates of heat capacity changes. By analyzing the distribution of changes in solvent-accessible surface area upon binding of FK506 to FKBP-12 from crystallographic data, it is found that 99% of the net surface buried upon binding involves nonpolar groups. This leads to a heat capacity change of FK506 binding, normalized to the amount of nonpolar surface, of -0.40 +/- 0.02 cal.K-1.mol-1.A-2 (1 cal = 4.18 J), a value similar to that obtained for the aqueous dissolution of hydrophobic substances. Our observations are discussed in view of the general nature of hydrophobic interaction processes.     

Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state

Protein-protein binding usually involves structural changes that may extend beyond the rearrangements on a local scale, and cannot be explained by a classical lock-and-key mechanism. Several models have been advanced to explain the flexible binding of proteins such as the induced fit mechanism where the ligand is postulated to induce a conformational change at the interaction site upon binding, or the preexisting equilibrium hypothesis that assumes that protein samples an ensemble of conformations at equilibrium conditions and that the ligand binds selectively to an active conformation. We explored the equilibrium motions of proteins that exhibit relatively large (nonlocal) conformational changes upon protein binding using the Gaussian network model and the anisotropic network model of protein dynamics. For four complexes, LIR-1/HLA-A2, Actin/DNase I, CDK2/cyclin, and CDK6/p16(INK4a), the motions calculated for the monomer exhibiting the largest conformational change, in its unbound (free) form, correlate with the experimentally observed structural changes upon binding. This study emphasizes the preexisting equilibrium/conformational selection as a mechanism for protein-protein interaction and lends support the concept that proteins, in their native conformation, are predisposed to undergo conformational fluctuations that are relevant to, or even required for, their biological functions.     

Atomic-scale dynamical structures of fatty acid bilayers observed by ultrafast electron crystallography

The structure and dynamics of a biological model bilayer are reported with atomic-scale resolution by using ultrafast electron crystallography. The bilayer was deposited as a Langmuir-Blodgett structure of arachidic (eicosanoic) fatty acids with the two chains containing 40 carbon atoms (approximately = 50 angstroms), on a hydrophobic substrate, the hydrogen terminated silicon(111) surface. We determined the structure of the 2D assembly, establishing the orientation of the chains and the subunit cell of the CH2 distances: a0 = 4.7 angstroms, b0 = 8.0 angstroms, and c0 = 2.54 angstroms. For structural dynamics, the diffraction frames were taken every 1 picosecond after a femtosecond temperature jump. The observed motions, with sub-angstroms resolution and monolayer sensitivity, clearly indicate the coherent anisotropic expansion of the bilayer solely along the aliphatic chains, followed by nonequilibrium contraction and restructuring at longer times. This motion is indicative of a nonlinear behavior among the anharmonically coupled bonds on the ultrashort time scale and energy redistribution and diffusion on the longer time scale. The ability to observe such atomic motions of complex structures and at interfaces is a significant leap forward for the determination of macromolecular dynamical structures by using ultrafast electron crystallography.     

How fast is protein hydrophobic collapse?

One of the most recurring questions in protein folding refers to the interplay between formation of secondary structure and hydrophobic collapse. In contrast with secondary structure, it is hard to isolate hydrophobic collapse from other folding events. We have directly measured the dynamics of protein hydrophobic collapse in the absence of competing processes. Collapse was triggered with laser-induced temperature jumps in the acid-denatured form of a simple protein and monitored by fluorescence resonance energy transfer between probes placed at the protein ends. The relaxation time for hydrophobic collapse is only approximately equal to 60 ns at 305 K, even faster than secondary structure formation. At higher temperatures, as the protein becomes increasingly compact by a stronger hydrophobic force, we observe a slowdown of the dynamics of collapse. This dynamic hydrophobic effect is a high-temperature analogue of the dynamic glass transition predicted by theory. Our results indicate that in physiological conditions many proteins will initiate folding by collapsing to an unstructured globule. Local motions will presumably drive the following search for native structure in the collapsed globule.     

Solvent fluctuations in hydrophobic cavity–ligand binding kinetics

Water plays a crucial part in virtually all protein–ligand binding processes in and out of equilibrium. Here, we investigate the role of water in the binding kinetics of a ligand to a prototypical hydrophobic pocket by explicit-water molecular dynamics (MD) simulations and implicit diffusional approaches. The concave pocket in the unbound state exhibits wet/dry hydration oscillations whose magnitude and time scale are significantly amplified by the approaching ligand. In turn, the ligand’s stochastic motion intimately couples to the slow hydration fluctuations, leading to a sixfold-enhanced friction in the vicinity of the pocket entrance. The increased friction considerably decelerates association in the otherwise barrierless system, indicating the importance of molecular-scale hydrodynamic effects in cavity–ligand binding arising due to capillary fluctuations. We derive and analyze the diffusivity profile and show that the mean first passage time distribution from the MD simulation can be accurately reproduced by a standard Brownian dynamics simulation if the appropriate position-dependent friction profile is included. However, long-time decays in the water–ligand (random) force autocorrelation demonstrate violation of the Markovian assumption, challenging standard diffusive approaches for rate prediction. Remarkably, the static friction profile derived from the force correlations strongly resembles the profile derived on the Markovian assumption apart from a simple shift in space, which can be rationalized by a time–space retardation in the ligand’s downhill dynamics toward the pocket. The observed spatiotemporal hydrodynamic coupling may be of biological importance providing the time needed for conformational receptor–ligand adjustments, typical of the induced-fit paradigm.     

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: from "protein quakes" to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 +/- 15 ns. at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.     

DNA motions in the nucleosome core particle.

We have used time-resolved triplet state anisotropy decay techniques to measure the conformational flexibility of DNA in the nucleosome. From these measurements we conclude that, in a nucleosome, the DNA helix experiences substantial internal flexibility, which occurs with a time constant near 30 nsec. We find that our data can be fit well by a modified version of the Barkley-Zimm model for DNA motion, allowing only DNA twisting motions and the overall tumbling of the nucleosome. That fit yields a calculated torsional rigidity equal to 1.8 X 10(-19) erg X cm, a value equal to that measured for uncomplexed DNA. We conclude from such similarity that large, fast twisting motions of the DNA helix persist, nearly unaltered, when DNA is wrapped to form a nucleosome.     

Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase

The hydrophobic effect--a rationalization of the insolubility of nonpolar molecules in water--is centrally important to biomolecular recognition. Despite extensive research devoted to the hydrophobic effect, its molecular mechanisms remain controversial, and there are still no reliably predictive models for its role in protein-ligand binding. Here we describe a particularly well-defined system of protein and ligands--carbonic anhydrase and a series of structurally homologous heterocyclic aromatic sulfonamides--that we use to characterize hydrophobic interactions thermodynamically and structurally. In binding to this structurally rigid protein, a set of ligands (also defined to be structurally rigid) shows the expected gain in binding free energy as hydrophobic surface area is added. Isothermal titration calorimetry demonstrates that enthalpy determines these increases in binding affinity, and that changes in the heat capacity of binding are negative. X-ray crystallography and molecular dynamics simulations are compatible with the proposal that the differences in binding between the homologous ligands stem from changes in the number and organization of water molecules localized in the active site in the bound complexes, rather than (or perhaps in addition to) release of structured water from the apposed hydrophobic surfaces. These results support the hypothesis that structured water molecules--including both the molecules of water displaced by the ligands and those reorganized upon ligand binding--determine the thermodynamics of binding of these ligands at the active site of the protein. Hydrophobic effects in various contexts have different structural and thermodynamic origins, although all may be manifestations of the differences in characteristics of bulk water and water close to hydrophobic surfaces.     

Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease

Niemann-Pick disease type C2 (NP-C2) is a fatal hereditary disease characterized by accumulation of low-density lipoprotein-derived cholesterol in lysosomes. Here we report the 1.7-A resolution crystal structure of the cholesterol-binding protein deficient in this disease, NPC2, and the characterization of its ligand binding properties. Human NPC2 binds the cholesterol analog dehydroergosterol with submicromolar affinity at both acidic and neutral pH. NPC2 has an Ig-like fold stabilized by three disulfide bonds. The structure of the bovine protein reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of approximately 160 A(3). We propose that this region represents the incipient cholesterol-binding site that dilates to accommodate an approximately 740-A(3) cholesterol molecule.     

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Despite their importance for biological activity, slower molecular motions beyond the nanosecond range remain poorly understood. We have assembled an unprecedented set of experimental NMR data, comprising up to 27 residual dipolar couplings per amino acid, to define the nature and amplitude of backbone motion in protein G using the Gaussian axial fluctuation model in three dimensions. Slower motions occur in the loops, and in the beta-sheet, and are absent in other regions of the molecule, including the alpha-helix. In the beta-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network of slow motion in the form of a standing wave extending across the beta-sheet, resulting in maximal conformational sampling at the interaction site. The alternating nodes along the sequence match the alternation of strongly hydrophobic side chains buried in the protein core. Confirmation of the motion is provided through extensive cross-validation and by independent hydrogen-bond scalar coupling analysis that shows this motion to be correlated. These observations strongly suggest that dynamical information can be transmitted across hydrogen bonds and have important implications for understanding collective motions and long-range information transfer in proteins.     

Dissection of complex protein dynamics in human thioredoxin

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8-13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10-17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.     

Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state (2)H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific (2)H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state (2)H NMR relaxation (spin-lattice, T(1Z), and quadrupolar-order, T(1Q)) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation--even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the (2)H NMR data suggest the β-ionone ring remains in its hydrophobic binding pocket in all three states of the protein. We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the β-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response--rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.     

Protein surface hydration mapped by site-specific mutations

Water motion at protein surfaces is fundamental to protein structure, stability, dynamics, and function. By using intrinsic tryptophans as local optical probes, and with femtosecond resolution, it is possible to probe surface-water motions in the hydration layer. Here, we report our studies of local hydration dynamics at the surface of the enzyme Staphylococcus nuclease using site-specific mutations. From these studies of the WT and four related mutants, which change local charge distribution and structure, we are able to ascertain the contribution to solvation by protein side chains as relatively insignificant. We determined the time scales of hydration to be 3-5 ps and 100-150 ps. The former is the result of local librational/rotational motions of water near the surface; the latter is a direct measure of surface hydration assisted by fluctuations of the protein. Experimentally, these hydration dynamics of the WT and the four mutants are also consistent with results of the total dynamic Stokes shifts and fluorescence emission maxima and are correlated with their local charge distribution and structure. We discuss the role of protein fluctuation on the time scale of labile hydration and suggest reexamination of recent molecular dynamics simulations.