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1.
A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.  相似文献   

2.
Specialized water-suppression NMR pulse sequences and heteronuclear three-dimensional NMR have now made it possible to observe cross relaxation between the protons of water molecules and protons on the surface or in the interior of biological molecules. The cross-relaxation characteristics at each site allow loosely and tightly bound waters to be distinguised and limits on the residence lifetimes to be placed. These observations are affording a new view of the nature of protein hydration.  相似文献   

3.
BackgroundAt initial steps of rehydration from cryptobiosis of anhydrobiotic organisms or at rehydration of dry tissues the liquid 1H NMR signal increased anomaly. The surplus in liquid signal may appear if some solid constituents dissolved, or if they were decomposed by enzymatic action.MethodsHydration kinetics, sorption isotherm, 1H NMR spectra and high power relaxometry were applied to monitor gaseous phase rehydration of Antarctic lichen Cetraria aculeata. Tightly and loosely bound water signal were distinguished, and the upper hydration limit for dissolution of water soluble solid fraction was not observed. A simple theoretical model was proposed.ResultsThe hydration courses showed a very tightly bound water fraction, a tightly bound water, and a loosely bound water fraction. Sigmoidal in form sorption isotherm was fitted well by multilayer sorption model. 1H NMR showed one Gaussian signal component from solid matrix of thallus and one or two Lorentzian line components from tightly bound, and from loosely bound water. The hydration dependency of liquid signal was fitted by rational function.ConclusionsAlthough in dehydrated C.aculeata the level of carbohydrates and polyols was low, the lichenase action during rehydration process increased it; the averaged saturation concentration cs=(57.3±12.0)%, which resembled that for sucrose.General significanceThe proposed method of water soluble solid fraction saturation concentration, cs, calculation from 1H NMR data may be applied for other organisms experiencing extreme dehydration or for dry tissues. We recalculated the published elsewhere data for horse chestnut (Aesculus hippocastanum) bast [water-soluble solid fraction recognized as sucrose, cs=(74.5±5.1)%]; and for Usnea antarctica, where cs=0.81±0.04.  相似文献   

4.
Initial stages of lichen hydration observed by proton magnetic relaxation   总被引:1,自引:0,他引:1  
The hydration of selected lichens ( Cladonia mitis , Cladonia bellidiflora , Cetraria islandica , Parmelia saxatilis , and Xanthoria parietina ) was investigated using gravimetry and proton magnetic free induction decays (FIDs).
The hydration from gaseous phase and dehydration to gaseous phase showed first-order kinetics. The amount of water which was non-removable in the air-dry state (relative humidity p / p 0=9%) did not depend significantly on the lichen species and was found to be 5·6±1·0% of the d. wt.
The proton FID Gaussian component from the solid matrix of thallus structure, and two (or, depending on lichen species, one averaged) liquid signals coming from water tightly bound on the surface of thallus solid matrix and from loosely bound or free water, were recorded. The bound-water component was distinguished by its motional properties and by its proximity to endogenous paramagnetic centres present in solid matrix (presumably PS II reaction centres of the photobiont). Mild dehydration (from gaseous phase) could completely remove the loosely bound water fraction, leaving the system below the water percolation threshold and below the water clustering point, emphasizing the passivity of lichen response to desiccation shock. In the species in which the one average liquid component was recorded, bound water behaved similarly.
The hydration at which free water pool vanishes (Δ M / m 0) and the relative (scaled to water) proton densities of solid matrix of lichen (β) were evaluated for all lichens investigated.  相似文献   

5.
Free polyribosomes and polyribosomes bound to endoplasmic membranes were isolated from 10-day-old chick embryos by differential centrifugation. The tightly and loosely bound polyribosomal fractions were isolated from the membrane-bound polyribosomes using 0,5 M KCl. The synthesis of collagen and non-collagen proteins on the polyribosomes were studied in a homologous cell-free system. It was shown that the polyribosomes tightly bound to the membranes possess a lower protein-synthesizing activity as compared to free and loosely bound polyribosomes. The amount of bacterial collagenase-cleaved polypeptides in the protein product synthesized on the polyribosomes tightly and loosely bound to the memranes and on free polyribosomes is 31, 23 and 9%, respectively. The data obtained suggest that the loosely bound polyribosomes are actively involved in collagen synthesis and that this fraction is not a contamination of free polyribosomes in the preparations of totally bound polyribosomes. The role of tightly and loosely bound polyribosomes in the formation of the membrane polyribosomal complex is discussed.  相似文献   

6.
Oxygen-17 and deuterium NMR studies of lysozyme hydration are reported for a wide range of lysozyme concentrations, and the relationship between water "activity" and water mobility in the lysozyme-water system as determined by high-field NMR is examined. In a first approximation, the effect of lysozyme activity on hydration is considered to be small because of the relatively low charge on lysozyme at pH 7 and the absence of salt in the lysozyme solutions. Correlation times are determined for tightly bound water, weakly bound water, and "multilayer" or trapped water in lysozyme at 20 degrees C. Hydration numbers are also determined for these three different water populations interacting with lysozyme. Good agreement is found between the hydration numbers determined by 17O NMR and the calculations based on the D'Arcy and Watt analysis of water sorption isotherms for proteins that considered three major water populations in hydrated lysozyme. A molecular interpretation for the three components in the D'Arcy and Watt theory of sorption isotherms is also proposed on the basis of our NMR results. Previous proton NMR spin-echo results are shown to be consistent with our findings by 17O NMR and support the view that there are at least four regions of distinct hydration behavior of lysozyme which span the whole range from solutions to solid powders.  相似文献   

7.
Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of kcat/Km for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH2 to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH2 that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.  相似文献   

8.
Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T2) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T2 data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.  相似文献   

9.
Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.  相似文献   

10.
A novel deuterium ((2)H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen-deuterium (H-D) exchange. Using this methodology differences in the H-D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity (a(w)), the degree of H-D exchange increased rapidly with hydration (from anhydrous to a(w) 0.22). At a(w) 0.22, subtilisin powders hydrated in air were found to have reached an H-D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a(w)) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n-hexane showed a significant reduction in H-D exchange at all a(w) studied. Control experiments demonstrated that the reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower level of exchange in n-hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix.  相似文献   

11.
The micellization process of dodecyltrimethylammonium chloride (DTAC) and bromide (DTAB) was studied. Nuclear magnetic resonance method was used. The 1H NMR and 13C NMR spectra were taken at higher and lower concentrations than the critical micelle concentrations (CMC) of the compounds studied. Chemical shifts were analysed. The studies performed were prompted by earlier calorimetric measurements which showed that there were significant qualitative and quantitative differences in the micellization process of the compounds studied. Namely, DTAB micelle dissociation was found to be an endothermic process while that of DTAC was exothermic. The differences found must be the result of differentiated influence of bromide and chloride counterions on the micellization process, including the phenomenon of micelle hydration. The objective of the work was to check whether cationic surfactant counterions can influence the micelle hydration process. Indeed, DTAB and DTAC, as monomers, exhibit similar hydrophobic hydration, but DTAB micelles are more hydrated than DTAC ones. It seems that the differences found in micellization of both salts studied may be attributed to different physicochemical properties of bromide and chloride ions, such as their mobilities and radii of their hydrated forms. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes, that hold the first hydration shell tightly, or water disordering chaotropes, that hold water molecules in that shell loosely.  相似文献   

12.
Ruan B  Fisher KE  Alexander PA  Doroshko V  Bryan PN 《Biochemistry》2004,43(46):14539-14546
Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.  相似文献   

13.
Structural data produced by a 2-ns molecular dynamics (MD) simulation on Geobacillus alanine racemase (AlaR; PDB: 1SFT) was used to study hydration around the two AlaR active sites. AlaR is a crucial enzyme for bacterial cell wall biosynthesis. It has been shown previously that the potency of an inhibitor can be increased by incorporating a functional group or atom that displaces hydration sites close to the substrate binding pocket of its target enzyme. The complete linkage algorithm was used for cluster analysis of the active site water positions from 126 solvent configurations sampled at regular intervals from the 2-ns MD simulation. Crystal waters in the 1SFT X-ray structure occupy most of the tightly bound water sites that were discovered. We show here that tightly bound water sites can be identified by cluster analysis of MD-generated coordinates starting with data supplied by a single X-ray structure, and we predict a highly conserved hydration site close to the carboxyl oxygen of L-Ala substrate. This approach holds promise for accelerating the drug design process. We also discuss an analysis of the well-known notion of residence time and introduce a new measure called retention time.  相似文献   

14.
The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine in H2O/2H2O mixtures (9:1, v/v) were investigated with 2H-NMR. Comparison of the 2H2O spin lattice relaxation time (T1) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H2O systems. Two distinct hydration regions could be discerned in the T1 relaxation time profile. (1) A minimum of 11-16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of T1 approximately equal to 90 ms. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with T1 = 500 ms and hydration water with T1 = 80-120 ms. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.  相似文献   

15.
The heterogeneity of bound acetylcholine and synaptic vesicles   总被引:5,自引:0,他引:5  
Synaptic vesicles containing radioactive acetylcholine have been isolated from slices of Torpedo electric organ incubated with radioactive choline. The recently synthesized radioactive acetylcholine is preferentially removed from the vesicles by iso-osmotic gel filtration. There is therefore a small compartment of loosely bound recently synthesized acetylcholine within the monodisperse vesicle fraction. The specific radioactivity of this compartment correlates most closely with the ;free' acetylcholine of electric organ that is lost when the tissue is homogenized. Membrane-associated vesicles did not contain any particular enrichment of this compartment. On standing at 6 degrees C the loosely bound compartment stabilizes so that it survives iso-osmotic filtration. A study of this phenomenon revealed that it was proportional to the extent of the loss of tightly bound acetylcholine from the vesicles. Incubation with Ca(2+), at pH5.5, or partial hypo-osmotic shock, caused losses of tightly bound acetylcholine and proportional increases in the stabilization of loosely bound acetylcholine of vesicles. Incubation at 20 degrees C caused less loss of tightly bound, and less stabilization of loosely bound, acetylcholine. A theoretical treatment of these exchanges also shows that the random factors promoting loss of tightly bound acetylcholine are statistically correlated with those which cause stabilization of loosely bound acetylcholine. The reciprocal relationship between the exchanges is inconsistent with there being two distinct populations of vesicles, one containing recently synthesized, loosely bound acetylcholine and the other containing tightly bound acetylcholine. It is proposed that all the vesicles contain a core of tightly bound acetylcholine and a surface layer of loosely bound acetylcholine. The origin of the extravesicular acetylcholine and also of the acetylcholine released on stimulation is discussed in the light of these results.  相似文献   

16.
Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K(i)=2.3+/-0.2 microM at pH 7.0 and 25 degrees C. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 107.3 ppm by (13)C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the (13)C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK(a)<3. This is the first observation of oxyanion formation in a reversible subtilisin-inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK(a) of approximately 6.5 in the free enzyme and on a pK(a)<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol(-1). We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.  相似文献   

17.
The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.  相似文献   

18.
Methyl -cyclodextrin (MCD) increased the activity and enantioselectivity of lyophilized subtilisin suspended in dry THF and acetonitrile in two transesterification model reactions. These beneficial improvements were diminished by the addition of water, in contrast to the observation that water activates subtilisin lyophilized from buffer alone. For example, the initial rate for the S enantiomer in the transesterification of vinylbutyrate with (±)-1-phenylethanol (sec-phenethylalcohol) decreased ca. 4-fold and the enantioselectivity from 59 to 40 when 0.1% (v/v) of water was added to THF.  相似文献   

19.
A molecular model is proposed to explain water 1H NMR spin-lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well-separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.  相似文献   

20.
The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.  相似文献   

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