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1.
Philo JS 《Analytical biochemistry》2000,279(2):151-163
The time-derivative method for deriving the sedimentation coefficient distribution, g(s*), from sedimentation velocity data that was developed by Walter Stafford has many advantages and is now widely used. By fitting Gaussian functions to the g(s*) distribution both sedimentation and diffusion coefficients (and therefore molecular masses) for individual species can be obtained. However, some of the approximations used in these procedures limit the accuracy of the results. An alternative approach is proposed in which the dc/dt data are fitted rather than g(s*). This new approach gives improved accuracy, extends the range to sedimentation coefficients below 1 S, and enhances resolution of multiple species. For both approaches the peaks from individual species are broadened when the data cover too wide a time span, and this effect is explored and quantified. An alternative algorithm for calculating ?(s*) from the dc/dt curves is presented and discussed. Rather than first averaging the dc/dt data for individual scan pairs and then calculating ?(s*) from that average, the ?(s*) distributions are calculated for every scan pair and then subsequently averaged. This alternative procedure yields smaller error bars for g(s*) and somewhat greater accuracy for fitted hydrodynamic properties when the time span becomes large. 相似文献
2.
X-ray structures of cubic insulin crystals in high concentrations of glucose at different pH levels and temperatures have been refined to high resolution. We have identified one glucose-binding site near the N-terminus of the A-chain whose occupancy is pH dependent. The effects of reduced water activity on the ordered protein and solvent structures have been examined. Our analysis showed no notable conformational changes in the ordered protein structures or ordered solvent molecules near the protein surface, but the presence of glucose does have a significant effect on the overall density distribution of the bulk solvent in the solvent-accessible volume. We compared the structure of cubic insulin at room temperature and liquid-nitrogen temperature, under identical solvent conditions, using glucose as a cryoprotectant. In this case, we found that the average temperature factor of the protein is reduced and more water molecules can be identified, but there are no significant changes in the protein conformation. 相似文献
3.
Ramu Anandakrishnan Aleksander Drozdetski Ross?C. Walker Alexey?V. Onufriev 《Biophysical journal》2015,108(5):1153-1164
Adequate sampling of conformation space remains challenging in atomistic simulations, especially if the solvent is treated explicitly. Implicit-solvent simulations can speed up conformational sampling significantly. We compare the speed of conformational sampling between two commonly used methods of each class: the explicit-solvent particle mesh Ewald (PME) with TIP3P water model and a popular generalized Born (GB) implicit-solvent model, as implemented in the AMBER package. We systematically investigate small (dihedral angle flips in a protein), large (nucleosome tail collapse and DNA unwrapping), and mixed (folding of a miniprotein) conformational changes, with nominal simulation times ranging from nanoseconds to microseconds depending on system size. The speedups in conformational sampling for GB relative to PME simulations, are highly system- and problem-dependent. Where the simulation temperatures for PME and GB are the same, the corresponding speedups are approximately onefold (small conformational changes), between ∼1- and ∼100-fold (large changes), and approximately sevenfold (mixed case). The effects of temperature on speedup and free-energy landscapes, which may differ substantially between the solvent models, are discussed in detail for the case of miniprotein folding. In addition to speeding up conformational sampling, due to algorithmic differences, the implicit solvent model can be computationally faster for small systems or slower for large systems, depending on the number of solute and solvent atoms. For the conformational changes considered here, the combined speedups are approximately twofold, ∼1- to 60-fold, and ∼50-fold, respectively, in the low solvent viscosity regime afforded by the implicit solvent. For all the systems studied, 1) conformational sampling speedup increases as Langevin collision frequency (effective viscosity) decreases; and 2) conformational sampling speedup is mainly due to reduction in solvent viscosity rather than possible differences in free-energy landscapes between the solvent models. 相似文献
4.
Harding SE Schuck P Abdelhameed AS Adams G Kök MS Morris GA 《Methods (San Diego, Calif.)》2011,54(1):136-144
In 1962 H. Fujita (H. Fujita, Mathematical Theory of Sedimentation Analysis, Academic Press, New York, 1962) examined the possibility of transforming a quasi-continuous distribution g(s) of sedimentation coefficient s into a distribution f(M) of molecular weight M for linear polymers using the relation f(M)=g(s)·(ds/dM) and showed that this could be done if information about the relation between s and M is available from other sources. Fujita provided the transformation based on the scaling relation s=κ(s)M(0.5), where κ(s) is taken as a constant for that particular polymer and the exponent 0.5 essentially corresponds to a randomly coiled polymer under ideal conditions. This method has been successfully applied to mucus glycoproteins (S.E. Harding, Adv. Carbohyd. Chem. Biochem. 47 (1989) 345-381). We now describe an extension of the method to general conformation types via the scaling relation s=κM(b), where b=0.4-0.5 for a coil, ~0.15-0.2 for a rod and ~0.67 for a sphere. We give examples of distributions f(M) versus M obtained for polysaccharides from SEDFIT derived least squares g(s) versus s profiles (P. Schuck, Biophys. J. 78 (2000) 1606-1619) and the analytical derivative for ds/dM performed with Microcal ORIGIN. We also describe a more direct route from a direct numerical solution of the integral equation describing the molecular weight distribution problem. Both routes give identical distributions although the latter offers the advantage of being incorporated completely within SEDFIT. The method currently assumes that solutions behave ideally: sedimentation velocity has the major advantage over sedimentation equilibrium in that concentrations less than 0.2mg/ml can be employed, and for many systems non-ideality effects can be reasonably ignored. For large, non-globular polymer systems, diffusive contributions are also likely to be small. 相似文献
5.
The solubility properties of paramyosin in the zones of pH and ionic strength in which aggregation occurs were initially studied using preparations isolated by a method originally described by Bailey (Bailey, K. (1956), Pubbl. Stn. Zool. Napoli 29, 26). Other preparations yielding apparently different protein components have been described by Hodge (Hodge, A.J. (1952), Proc. Natl. Acad. Sci., U.S.A. 38, 850) using acid conditions, and Stafford and Yphantis (Stafford, W.F., AND Yphantis, D. (1972), Biochem. Biophys. Res. Commun. 49, 848) have identified alpha-, beta-, and gamma-paramyosin using various times and temperatures of extraction with or without ethylenediaminetetraacetic acid. We have found that acid-extracted paramyosin is very similar if not identical to alpha-paramyosin, but that both acid and alpha forms differ considerably from beta- and gamma-paramyosin. Beta-Paramyosin precipitates abruptly from solution in narrow zone of pH below neutrality, and increases in ionic strength shift the zone of precipitation toward lower pH values. In contrast, both acid and alpha-paramyosin show gradual aggregation with changing pH at lowerionic strength (less than 0.3) but sharp transitions similar to beta-paramyosin at higher ionic strength (greater than 0.3). Transitions were also found at lower pH (ca. 4.0) which were not mirror images of transitions at higher pH (ca. 7.0). Viscosity measurements show that acid extracted paramyosin is close in behavior to a native extract obtained by extraction in mild, nondenaturing media containing mixed antibiotics. Each of these extracts differed considerably from beta-paramyosin. Mild, nonhydrolytic procedures employed by others to remove small, noncovalent bonded components or to separate protein complexes were not effective in converting alpha- to beta-paramyosin. Comparison of extraction procedures strongly supports the suggestion of Stafford and Yphantis that beta- and gamma-paramyosin are hydrolytic products of alpha-paramyosin and that the proteases responsible may be of bacterial origin. 相似文献
6.
P J Steinbach A Ansari J Berendzen D Braunstein K Chu B R Cowen D Ehrenstein H Frauenfelder J B Johnson D C Lamb 《Biochemistry》1991,30(16):3988-4001
Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme. 相似文献
7.
Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism. 相似文献
8.
The same primary structure of the prion protein yields two distinct self-propagating states 总被引:2,自引:0,他引:2
The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptide-based biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrP(Sc)-specified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in self-propagating protein states underlies prion strain diversity. 相似文献
9.
R B Gregory 《Biopolymers》1988,27(11):1699-1709
Hydrogen isotope exchange rates for lysozyme in glycerol cosolvent mixtures [D. G. Knox and A. Rosenberg (1980) Biopolymers 19 , 1049–1068] have been analyzed as functions of solvent viscosity and glycerol activity in an attempt to determine which solvent properties influence protein internal dynamics. The effect of glycerol on the fast- and slow-exchanging protons is different. Slow-exchanging protons [H(t) < 20] are slowed by ever-increasing amounts as H(t) decreases. However, comparison with data for the effect of glycerol on the thermal unfolding of lysozyme [K. Gekko (1982) J. Biochem. 19 , 1197–1204] indicates that the large decrease in exchange rates for the slow protons is not consistent with a local unfolding mechanism of exchange. These effects are also too large to be easily rationalized in terms of solvent viscosity. Instead, we suggest that the large effect of glycerol on exchange of the slow protons is due to a “compression” of the protein, as a result of thermodynamically unfavorable interactions of glycerol with the protein surface. This reduces the protein void volume, which in turn decreases the probability of conformational transitions required for exchange of the slowest protons. Present data do not allow a distinction to be made between thermodynamic (glycerol activity) and dynamic (solvent viscosity) influences on exchange rates for the fast-exchanging protons, although the effect of glycerol on these protons is also probably too large to be consistent with a local unfolding mechanism. In this case, glycerol decreases the rate of catalyst diffusion within the protein matrix, either by decreasing the probabilities or amplitudes of “gating” reactions that allow passage of the catalyst from the solvent to the exchange site, or by increasing the relaxation times for these conformational rearrangements. 相似文献
10.
The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates. 相似文献
11.
Reinhard Schweitzer‐Stenner Andrew Hagarman Siobhan Toal Daniel Mathieu Harald Schwalbe 《Proteins》2013,81(6):955-967
We performed a conformational analysis of the central residues of three tripeptides glycyl‐L ‐isoleucyl‐glycine (GIG), glycyl‐L ‐tyrosyl‐glycine (GYG) and glycyl‐L ‐arginyl‐glycine (GRG) in aqueous solution, based on a global analysis of amide I′ band profiles and NMR J‐coupling constants. The results are compared with recently reported distributions of GVG, GFG and GEG. For GIG and GYG, we found that even though the polyproline II (pPII) fraction is below 0.5, it is still the most populated conformation, whereas GVG and GFG show both a larger β‐strand fraction. For GRG, we observed a clear dominance of pPII over β‐strand, reminiscent of observations for GEG and GKG. This finding indicates that terminal charges on otherwise hydrophobic residue side chains stabilize pPII over β‐strand conformations. For all peptides investigated we found that a variety of compact and turn‐like conformations constitute nearly 20 percent of their conformational distributions. Attempts to analyze our data with a simple two‐state pPII??β model therefore do not yield any satisfactory reproduction of experimental results. A comparison of the obtained GxG ensembles with conformational distributions of GxG segments in truncated coil libraries (helices and sheets omitted) revealed a much larger fraction of type II βi+2 and type III β like conformations for the latter. Thus, a comparison of conformational distributions of unfolded peptide segments in solution and in coil libraries reveal interesting information on how the interplay between intrinsic propensities of amino acid residues and non‐local interactions in polypeptide chains determine the conformations of loop segments in proteins. Proteins 2013; © 2012 Wiley Periodicals, Inc. 相似文献
12.
Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions. 相似文献
13.
Sedimentation velocity experiments can be used to identify two or more independent non-interacting macromolecules, which differ
in their size by only a few percent. The procedure requires the extrapolation of differential apparent sedimentation coefficient
distributions obtained at different running time to t → ∞ and works because it eliminates or greatly reduces diffusion effects.
Here, we present an improved time extrapolation function of sedimentation distribution profiles originally presented by Stafford
(In: Harding, Rowe, Horton (eds.) Analytical ultracentrifugation in biochemistry and polymer science, 1992). We describe a computing procedure with the program lamm to analyze concentration profiles obtained by absorbance or interference optics that utilizes suitable smoothing methods
for noisy data sets and present examples which include time invariant noises. 相似文献
14.
Validating Solution Ensembles from Molecular Dynamics Simulation by Wide-Angle X-ray Scattering Data
Wide-angle x-ray scattering (WAXS) experiments of biomolecules in solution have become increasingly popular because of technical advances in light sources and detectors. However, the structural interpretation of WAXS profiles is problematic, partly because accurate calculations of WAXS profiles from structural models have remained challenging. In this work, we present the calculation of WAXS profiles from explicit-solvent molecular dynamics (MD) simulations of five different proteins. Using only a single fitting parameter that accounts for experimental uncertainties because of the buffer subtraction and dark currents, we find excellent agreement to experimental profiles both at small and wide angles. Because explicit solvation eliminates free parameters associated with the solvation layer or the excluded solvent, which would require fitting to experimental data, we minimize the risk of overfitting. We further find that the influence from water models and protein force fields on calculated profiles are insignificant up to q ≈ 15 nm?1. Using a series of simulations that allow increasing flexibility of the proteins, we show that incorporating thermal fluctuations into the calculations significantly improves agreement with experimental data, demonstrating the importance of protein dynamics in the interpretation of WAXS profiles. In addition, free MD simulations up to one microsecond suggest that the calculated profiles are highly sensitive with respect to minor conformational rearrangements of proteins, such as an increased flexibility of a loop or an increase of the radius of gyration by < 1%. The present study suggests that quantitative comparison between MD simulations and experimental WAXS profiles emerges as an accurate tool to validate solution ensembles of biomolecules. 相似文献
15.
Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
17.
Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents
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We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle. 相似文献
18.
J. Alejandro D'Aquino Javier Gmez Vincent J. Hilser Kon Ho Lee L. Mario Amzel Ernesto Freire 《Proteins》1996,25(2):143-156
The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent-exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (ΔCp), identical solvent-related entropies of unfolding (ΔSsolv), and identical enthalpies of unfolding (ΔH) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles ϕ and ϱ. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 ± 0.2 cal/K · mol with respect to that of glycine, consistent with a 3.4-fold reduction in the number of allowed conformations in the alanine-containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position β. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K · mol in the backbone entropy was obtained for leucine and lysine, respectively. β-branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K · mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an α-helix is 6.51 cal/K · mol for glycine. These and previous results have allowed a complete estimation of the conformational entropy changes associated with protein folding. © 1996 Wiley-Liss, Inc. 相似文献
19.
Hans Frauenfelder 《Journal of biological physics》2005,31(3-4):413-416
Proteins are not isolated homogeneous systems. Each protein can exist in a very large number of conformations (conformational
substates) that are characterized by an energy landscape. The main conformational motions, similar to the α and β fluctuations
in glasses, are linked to fluctuations in the bulk solvent and the hydration shell. 相似文献
20.
Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.
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M Graupner L Haalck F Spener H Lindner O Glatter F Paltauf A Hermetter 《Biophysical journal》1999,77(1):493-504
We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol. 相似文献