Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants

OBJECTIVES: To evaluate the effectiveness and morbidity of salvage brachytherapy for locally recurrent or persistent prostate cancer after radiotherapy failure. METHODS: In this retrospective study, 49 patients of median age 73.3 years (range 52.9 to 86.9) with biopsy-proven localized prostate cancer underwent interactive transperineal fluoroscopic-guided and biplane ultrasound-guided brachytherapy with either iodine 125 or palladium 103 after prior radiotherapy failure. Post-treatment follow-up was conducted for a median of 64.1 months (range 26.6 to 96.8) and included clinical assessment of disease status, assays of serum prostate-specific antigen (PSA) levels, and documentation of treatment-related symptoms and complications. Determination of biochemical treatment failure was based on two successive rising PSA values above the post-treatment PSA nadir value. RESULTS: The actuarial rate of local prostate cancer control was 98% (95% confidence interval [CI] 94% to 99%). Actuarial disease-specific survival at 3 and 5 years was 89% (95% CI 73% to 96%) and 79% (95% CI 58% to 91%), respectively. At 3 and 5 years, actuarial biochemical disease-free survival was 48% (95% CI 32% to 63%) and 34% (95% CI 17% to 51%), respectively. Post-treatment PSA nadir was found to be a significant predictor of biochemical disease-free survival. Actuarial biochemical disease-free survival of patients who achieved a PSA nadir less than 0.5 ng/mL was 77% (95% CI 53% to 90%) and 56% (95% CI 25% to 78%) at 3 and 5 years, respectively. Of 49 patients, 23 (47%) achieved a post-treatment PSA nadir less than 0.5 ng/mL. The incidence of serious complications after salvage brachytherapy, such as incontinence and rectal complications, was lower than that reported after other types of salvage procedures. CONCLUSIONS: Interactive transperineal fluoroscopic-guided and biplane ultrasound-guided brachytherapy is a novel, potentially curative salvage modality for patients in whom prior radiotherapy failed. In a population of patients with poor prognosis, this modality was associated with a high rate of local prostate cancer control and a 34% actuarial rate of biochemical disease-free survival at 5 years. The incidence of major complications after salvage brachytherapy appears to be lower than that after other potentially curative salvage procedures, such as radical prostatectomy and cryoablation. Salvage brachytherapy warrants further investigation.     

Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites

A lexical decision experiment investigated hemisphere asymmetries in resolving lexical ambiguity within a sentence context. Sentences that biased a single meaning (either dominant or subordinate) of sentence-final ambiguous words were followed by a lateralized target related to the sentence-congruent or -incongruent meaning of the ambiguous word, or an unrelated word. In the RVF sentence-congruent targets were facilitated, while incongruent targets were not primed. In contrast, related targets were facilitated in the LVF, regardless of sentence context. This suggests that selecting the contextually appropriate word meaning requires the left hemisphere, and supports a right hemisphere role in maintaining alternate word senses.     

DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: analysis with mutated binding sites

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5' or 3' sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.     

Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina

The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus.     

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.     

Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid-binding proteins

We constructed 18 single amino acid mutants of the adipocyte fatty acid-binding protein (A-FABP) and 17 of the intestinal fatty acid-binding protein (I-FABP), at locations in the fatty acid (FA) binding sites. For each mutant protein, we measured thermodynamic parameters that characterize FA binding. Binding affinities ranged from about 200-fold smaller to 30-fold larger than the wild type (WT) proteins. Thermodynamic parameters revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory and larger than the binding free energy. FA-FABP interactions were quite different for I-FABP and A-FABP proteins. Binding affinities were larger and decreased to a greater degree with increasing FA solubility for most of the I-FABP as compared with the A-FABP proteins, consistent with a more hydrophobic binding site for the I-FABP proteins. In A-FABP, Ala substitutions for Arg106 and Arg126, which interact with the FA carboxylate, reduce affinities by about 100-fold, but in I-FABP, R106A increases affinities up to 30-fold. Moreover, in A-FABP, the thermodynamic parameters predict that the FA carboxylate location switches from the 126-position in R106A to the 106 position in R126A. Finally, the A-FABP proteins, in contrast to the I-FABP proteins, reveal significant heat capacity changes (DeltaCp) upon FA binding, and substitutions at residues Arg106 and Arg126 reduce the magnitude of DeltaCp.     

Carnitine palmitoyltransferase activities: effects of serum albumin, acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low microM concentrations of octanoyl-CoA. However, even a large excess (500 microM) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low microM concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above affect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolism in vivo.     

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.     

Endothelial binding sites for lipoprotein lipase are not diminished in perfused hearts from diabetic rats

The possibility that diabetes reduces functional, heparin-releasable lipoprotein lipase (HR-LPL) activity on the coronary vasculature of perfused hearts by altering endothelial binding sites for the enzyme was examined by measuring the binding and subsequent heparin-induced release of exogenous lipoprotein lipase purified from bovine milk (mLPL). Rat hearts were first perfused with heparin (5 U/mL) for 5 min to displace endogenous HR-LPL into the perfusate. The subsequent perfusion of control hearts with 0.05-2 micrograms/mL mLPL resulted in a progressive increase in bound exogenous enzyme that could be released by a second heparin perfusion. Induction of an acute, insulin-deficient model of diabetes (100 mg/kg streptozotocin 4-5 days prior to heart perfusions) reduced endogenous HR-LPL activity, but the binding and heparin-induced release of mLPL (0.5 microgram/mL) were the same as measured in control hearts. Therefore, diabetes does not alter low-affinity, high-capacity proteoglycan binding sites for mLPL on the endothelium of perfused hearts.     

Remarks on the phylogeny and structure of fatty acid binding proteins from parasitic platyhelminths

Four fatty acid binding proteins (FABPs) have been described in 4 parasitic platyhelminths: Schistosoma mansoni, Schistosoma japonicum, Fasciola hepatica and Echinococcus granulosus. FABPs form a multigenic family of cytosolic proteins widely distributed in metazoan tissues, the function of which is still poorly understood. These helminth proteins have recently received attention, since there are reports to indicate that S. mansoni and F. hepatica FABPs may be protective antigens. In addition, these proteins could play a major role in the parasites' life-cycles because platyhelminths are unable to synthesize de novo most of their lipids. We have undertaken phylogenetic and structural analyses of platyhelminth FABPs in an attempt to characterize features of biological relevance. Phylogenetically, these FABPs appear to be more closely related to those of vertebrate heart, mammary gland, muscle, retina, skin, brain and myelin, although no clear functional relationships were established between them. We describe several conserved motifs characteristic of specific groups of FABPs. Hydrophilicity, flexibility and accessibility analyses revealed several major putative epitopes for the E. granulosus FABP, EgDf1, that appear to be centred in loops of the EgDf1 3-dimensional structure modelled by molecular replacement.     

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.     

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.     

Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli

Tryptophanyl-tRNA synthetase of Escherichia coli has 1.8 binding sites for L-tryptophan with Kdiss of 12 x 10(-5) M as shown by equilibrium dialysis. The results are in accord with the known structure of the enzyme, and alpha2 dimer of 74,000 molecular weight, and with 2 binding sites for tryptophanyl-ATP ester. Ordinary sucrose density gradient centrifugation reveals a complex composed of one tRNATrp bound per enzyme dimer. When tRNATrp is mixed throughout the gradient at concentrations from 5.4 x 10(-6) M to 2.0 x 10(-5) M, a new peak appears in the position expected for a complex with two tRNATrp molecules bound per enzyme dimer. Sedimentation through gradients lacking tRNATrp favors dissociation of the 1:2 complex but not the 1:1 complex. The data indicate 2 binding sites for tRNATrp on tryptophanyl-tRNA synthetase.     

Defining the arachidonic acid binding site of human 15-lipoxygenase. Molecular modeling and mutagenesis

Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.     

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.     

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].     

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.     

Water molecules in the binding cavity of intestinal fatty acid binding protein: dynamic characterization by water 17O and 2H magnetic relaxation dispersion

The hydration of intestinal fatty acid binding protein (IFABP) in apo-form and complexed with palmitate, oleate, and 1-anilino-8-naphthalene sulfonate (ANS) has been studied by water 17O and 2H magnetic relaxation dispersion (MRD) measurements. These ligands bind in a large internal cavity, displacing most of the crystallographically identified cavity water molecules. Unlike most other proteins, IFABP gives rise to MRD profiles with two dispersion steps. The low-frequency dispersion yields a correlation time of 7 ns at 300 K, matching the known tumbling time of IFABP. The dispersion amplitude requires only three (apo) or four (holo) long-lived and ordered water molecules (residence time 0.01-4 microseconds at 300 K). Comparison of MRD profiles from the different complexes indicates that the displaced cavity water molecules are short-lived. The few long-lived (>10 ns) water molecules required by the MRD data are tentatively assigned to crystallographic hydration sites on the basis of accessibility, positional order, and H-bonding. The amplitude of the high-frequency dispersion corresponds to 10-20 moderately ordered water molecules, with a correlation time of ca. 1 ns that may reflect a transient opening of the cavity required for exchange with external water.