Thermal denaturation of ribonuclease A characterized by water 17O and 2H magnetic relaxation dispersion

Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (NMRD) measurements were used to characterize ribonuclease A (RNase A) in the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water molecules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least three water molecules with a mean residence time of 8 ns at 27 degreesC and an activation enthalpy of ca. 40 kJ mol-1. These water molecules are identified with some or all of six ordered water molecules partly buried in surface pockets in the crystal structure of RNase A. The loss of the 17O dispersion at higher temperatures demonstrates that, in the thermally denatured protein, these surface pockets are either not present or undergoing large structural fluctuations on a subnanosecond time scale. The relaxation dispersion step vanishes monotonically and essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amount of non-native and long-lived hydration water. The NMRD data show that thermally denatured RNase A has a relatively compact but highly flexible structure. The global solvent exposure and the hydrodynamic volume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturation is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatured state, these side chains sample a wide distribution of conformational states on a subnanosecond time scale.     

A new view of water dynamics in immobilized proteins

The inflection frequency of the deuteron magnetic relaxation dispersion from water in rotationally immobilized protein samples has recently been found to be essentially independent of temperature and protein structure. This remarkable invariance has been interpreted in terms of a universal residence time of 1 microseconds for protein-associated water molecules. We demonstrate here that this interpretation is an artifact of the conventional perturbation theory of spin relaxation, which is not valid for rotationally immobile proteins. Using a newly developed non-perturbative, stochastic theory of spin relaxation, we identify the apparent correlation time of 1 microseconds with the inverse of the nuclear quadrupole frequency, thus explaining its invariance. The observed dispersion profiles are consistent with a broad distribution of residence times, spanning the microseconds range. Furthermore, we argue that the deuteron dispersion is due to buried water molecules rather than to the traditional surface hydration previously invoked, and that the contribution from rapidly exchanging protein hydrogens cannot be neglected. The conclusions of the present work are also relevant to proton relaxation in immobilized protein samples and to magnetic resonance imaging of soft tissue.     

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.     

Determination of the residence time of water molecules hydrating B'- DNA and B-DNA, by one-dimensional zero-enhancement nuclear Overhauser effect spectroscopy

The residence time of water in the minor groove of the d(CGCGAATTCGCG) duplex has been determined by a recent measurement combining nuclear Overhauser enhancements (NOE, ROE) and 17O relaxation dispersion. The time is in the range of nanoseconds, so that it may be measured by a rather simple method proposed here, namely the choice of conditions such that the NOE between the observed DNA proton and a nearby water proton is zero. This condition is realized when the residence time of the water molecule is 0.178 times the nuclear magnetic resonance period (e.g. 0.297 ns at 600 MHz). It may be achieved by varying the magnetic field and/or the temperature. The zero-NOE measurement may be performed by one-dimensional NMR, and has therefore good sensitivity. We have developed excitation sequences which suppress two spurious contributions to the NOE: from neighboring exchangeable protons and from H3' protons whose chemical shift is close to that of water. The method is applied here to the comparison of residence times of water next to B-DNA and next to B'-DNA, the latter corresponding to better stacked, propeller-twisted base-pairs and a correspondingly narrower minor groove. In the minor groove of [d(CGCGAATTCGCG)]2, a B'-DNA duplex, the residence time of the water molecule next to H2 of adenine(6) (underlined), is 0.6 ns at 10 degreesC, in good agreement with the value obtained previously. The residence time is slightly but distinctly shorter for the water next to A5, suggesting non-cooperative departure of these two molecules which are presumed to be part of the hydration spine. Near A5 and A4 of [d(AAAAATTTTT)]2, another B'-DNA duplex, the residence times are approximately twice as long, but the activation enthalpies are about the same, ca. 38 kJ/mol. The residence time in the minor groove of the regular B-DNA sequence d(CGCGATCGCG) was 0.3 ns at 10 degreesC, shorter than in the case of the B'-DNA sequences by factors of 2 and 4, respectively. The temperature dependence is less, with an activation enthalpy of 27 kJ/mol. The major groove residence times are comparable for the three sequences, and a few times shorter than those of minor groove water. A value of 0.36 ns, or even more in case of rotation of water, is obtained around -8 degreesC. The most striking aspect of these results is the relatively small difference in the residence times of reputedly fast and slow-exchanging water molecules bound to DNA in biological conditions. This suggests that the spine of hydration is perhaps not a major stabilizer of the B'-DNA structure as compared with B-DNA.     

水化在18-冠醚-6对K+和Na+选择过程中作用的理论研究

A combination of molecular dynamics (MD) and density functional theory (DFT) calculations were used to study the hydration structures of K+ and Na+ ions under the confinement of 18-crown-6 in order to identify the role of water in the selectivity of 18-crown-6 towards K+. The radial distribution functions, coordination numbers, orientation structures and interaction energies were analyzed to investigate the hydration of K+ and Na+ in 18-crown-6/cation complexes. All calculations of K+ and Na+ in bulk water were also conducted for comparison.The simulation results show that the orientation distributions of the water molecules in the first coordination shell of K+ are more sensitive to the confinement of 18-crown-6 than those of Na+. It is more favorable to confine a K+ in 18-crown-6 than a Na+ in terms of interaction energy. Good agreement is obtained between MD results and DFT results.     

Biological therapy of ovarian cancer: current directions

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol-1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable.     

Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: a functional, spectroscopic, and molecular modeling study

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].     

Hydration structure of a collagen peptide

BACKGROUND: The collagen triple helix is a unique protein motif defined by the supercoiling of three polypeptide chains in a polyproline II conformation. It is a major domain of all collagen proteins and is also reported to exist in proteins with host defense function and in several membrane proteins. The triple-helical domain has distinctive properties. Collagen requires a high proportion of the post-translationally modified imino acid 4-hydroxyproline and water to stabilize its conformation and assembly. The crystal structure of a collagen-like peptide determined to 1.85 Angstrum showed that these two features may be related. RESULTS: A detailed analysis of the hydration structure of the collagen-like peptide is presented. The water molecules around the carbonyl and hydroxyprolyl groups show distinctive geometries. There are repetitive patterns of water bridges that link oxygen atoms within a single peptide chain, between different chains and between different triple helices. Overall, the water molecules are organized in a semi-clathrate-like structure that surrounds and interconnects triple helices in the crystal lattice. Hydroxyprolyl groups play a crucial role in the assembly. CONCLUSIONS: The roles of hydroxyproline and hydration are strongly interrelated in the structure of the collagen triple helix. The specific, repetitive water bridges observed in this structure buttress the triple-helical conformation. The extensively ordered hydration structure offers a good model for the interpretation of the experimental results on collagen stability and assembly.     

Hydration force in the atomic force microscope: A computational study

Using a hard sphere model and numerical calculations, the effect of the hydration force between a conical tip and a flat surface in the atomic force microscope (AFM) is examined. The numerical results show that the hydration force remains oscillatory, even down to a tip apex of a single water molecule, but its lateral extent is limited to a size of a few water molecules. In general, the contribution of the hydration force is relatively small, but, given the small imaging force ( approximately 0.1 nN) typically used for biological specimens, a layer of water molecules is likely to remain "bound" to the specimen surface. This water layer, between the tip and specimen, could act as a "lubricant" to reduce lateral force, and thus could be one of the reasons for the remarkably high resolution achieved with contact-mode AFM. To disrupt this layer, and to have a true tip-sample contact, a probe force of several nanonewtons would be required. The numerical results also show that the ultimate apex of the tip will determine the magnitude of the hydration force, but that the averaged hydration pressure is independent of the radius of curvature. This latter conclusion suggests that there should be no penalty for the use of sharper tips if hydration force is the dominant interaction between the tip and the specimen, which might be realizable under certain conditions. Furthermore, the calculated hydration energy near the specimen surface compares well with experimentally determined values with an atomic force microscope, providing further support to the validity of these calculations.     

Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.     

RNA hydration: a detailed look

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.     

The role of hydration in the hydrolysis of pyrophosphate. A Monte Carlo simulation with polarizable-type interaction potentials

The exchange of energy in biochemical reactions involves, in a majority of cases, the hydrolysis of phosphoanhydrides (P-O-P). This discovery has lead to a long discussion about the origin of the high energy of such bonds, and to a proposal that hydration plays a major role in the energetics of the hydrolysis. This idea was supported by recent ab initio quantum mechanical calculations (Saint-Martin et al. (1991) Biochim. Biophys. Acta 1080, 205-214) that predicted the hydrolysis of pyrophosphate is exothermic in the gas phase. This exothermicity can account for only a half of the total energy release that one measures in aqueous solutions. Here we address the problem of hydration of the reactants and products of the pyrophosphate hydrolysis by means of Monte Carlo simulations, employing polarizable potentials whose parameters are fitted to energy surfaces computed at the SCF/6-31G** level of the theory. The present results show that the hydration enthalpies of the reactants and products contribute significantly to the total energy output of the pyrophosphate hydrolysis. The study predicts that both, the orthophosphate and the pyrophosphate, have hydration spheres with the water molecules acting as proton acceptors in the P-OH ... O(water) hydrogen bonds. These water molecules weakly repel the water molecules in the further hydration spheres. The perturbation of the structure of the solvent caused by the presence of the solute molecules is short ranged: after ca. 5 A from the P atoms, the energy and the structure of water correspond to bulk water. Due mainly to nonadditive effects, the molecular structure of the hydrated pyrophosphate is quite different from two fused structures of the hydrated orthophosphates. The hydration sphere of pyrophosphate is very loose and has a limited effect on the water network, whereas for orthophosphate it has a well developed shell structure. Hence, upon hydration there will be both a gain in hydration enthalpy and a gain in entropy because of distortion of the water molecular network.     

Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.     

Thermodynamics of dT--dT base pair mismatching in linear DNA duplexes and three-arm DNA junctions

We have used a combination of magnetic-suspension densimetry and calorimetry to derive complete thermodynamic profiles, including volume changes, for the formation of linear DNA duplexes and three-arm branched DNA junctions, from their component strands, with and without dT-dT mismatches. The formation of each type of complex at 20 degrees C is accompanied by a favorable free energy, with a favorable enthalpy term partially compensated by an unfavorable entropy. Formation is associated also with net uptake of water molecules. Using the formation of the fully-paired linear duplex or three-arm junction as reference states, we can establish a thermodynamic cycle in which the contribution of the single-strand species cancels. From this cycle, we determine that substitution of dA for dT has a differential free energy of deltadeltaG degrees of +2.4 kcal mol(-1) for mismatched duplex and +2.0 kcal mol(-1) (on the average) for the mismatched junction. These unfavorable differential free energies result from an unfavorable enthalpy, partially compensated by a favorable entropy, and a negative deltadeltaV. The free energies in the two cases have signs opposed to those of deltadeltaV, a situation that implicates hydration changes in creating the mismatch. When the deltadeltaV terms are normalized by the total number of base pairs involved, the immobilization of structural water molecules (and/or substitution of electrostricted for hydrophobic water molecules) is about 7 times greater for junctions than duplexes. This is consistent with more extensive hydrophobic hydration of branched DNA structures than of duplexes.     

Multiple hydration layers in cubic insulin crystals

Cubic insulin crystals contain approximately 30-A-diameter channels filled with aqueous solvent, providing a useful system in which to analyze hydration structure at a variety of distances from protein surfaces. Beginning with an atomic model for the protein and ordered water molecules, the density distribution in the solvent volume of the phasing model was iteratively refined to improve the fit of calculated structure factors with x-ray diffraction data. The free R value, which compares calculated structure factors with a subset of observed structure factors deliberately omitted from the refinement, was used to provide an objective confirmation of the effectiveness of the refinement procedure. Electron density maps of the solvent, computed using the solvent-refined phases and complete low-resolution diffraction data, reveal multiple hydration layers around the protein.     

Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein

Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.     

1H NMR study of the structure and dynamics of water confined between wetting solids

1H NMR relaxation measurements in a broad range of frequencies give information on the dynamical behaviour of water molecules in dilute suspensions of swelling clay (Hectorite). The logarithmic frequency variation of the longitudinal relaxation rate suggests a 2D diffusion of the water molecules adsorbed on the clay particles, with a residence time as long as 10 6 s. The corresponding high activation free energy (40 kJ/mol) originates mainly from the clay-water interactions, as shown by Monte Carlo simulations of clay hydration.     

The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.     

Early self-experienced neuropsychological deficits and subsequent schizophrenic diseases: an 8-year average follow-up prospective study

Magnetization transfer through dipole-dipole interactions (nuclear Overhauser effects, NOEs) between water protons and the protons lining two small hydrophobic cavities in hen egg-white lysozyme demonstrates the presence of water molecules with occupancies of approximately 10-50%. Similarly, NOEs were observed between the cavity protons and the protons of hydrogen, methane, ethylene or cyclopropane applied at 1-200 bar pressure. These gases can thus be used as general NMR indicators of empty or partially hydrated hydrophobic cavities in proteins. All gases reside in the cavities for longer than 1 ns in marked contrast to common belief that gas diffusion in proteins is not much slower than in water. Binding to otherwise empty cavities may be a major aspect of the anesthetic effect of small organic gas molecules.