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1.
Fluorescence studies of the conformational dynamics of parvalbumin in solution: lifetime and rotational motions of the single tryptophan residue 总被引:1,自引:0,他引:1
S T Ferreira 《Biochemistry》1989,28(26):10066-10072
The fluorescence properties of the single tryptophan residue in whiting parvalbumin were used to probe the dynamics of the protein matrix. Ca2+ binding caused a blue-shift in the emission (from lambda max = 339 to 315 nm) and a 2.5-fold increase in quantum yield. The fluorescence decay was nonexponential in both Ca2(+)-free and Ca2(+)-bound parvalbumin and was best described by Lorentzian lifetime distributions centered around two components: a major long-lived component at 2-5 ns and a small subnanosecond component. Raising the temperature from 8 to 45 degrees C resulted in a decrease in both the center (average) and width (dispersion) of the major lifetime distribution component, whereas the center, width, and fractional intensity of the fast component increased with temperature. Arrhenius activation energies of 1.3 and 0.3 kcal/mol were obtained in the absence and in the presence of Ca2+, respectively, from the temperature dependence of the center of the major lifetime distribution component. Direct anisotropy decay measurements of local tryptophan rotations yielded an activation energy of 2.3 kcal/mol in Ca2(+)-depleted parvalbumin and indicated a correlation between rotational rates and lifetime distribution parameters (center and width). Ca2+ binding produced a decrease in the width of the major lifetime distribution component and a decrease in tryptophan rotational mobility within the protein. There was a rough correlation between these two parameters with changes in Ca2+ and temperature, so that both measurements may be taken to indicate that the structure of Ca2(+)-bound parvalbumin was more rigid than in Ca2(+)-depleted parvalbumin. 相似文献
2.
Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature. 相似文献
3.
Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy 总被引:1,自引:0,他引:1
Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence. 总被引:4,自引:1,他引:3
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Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein. 相似文献
5.
This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins. 相似文献
6.
The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods. In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark [D] + [E] slow passage ODMR signal. These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes. The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction. Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms. The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s). This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues. 相似文献
7.
The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites. 相似文献
8.
Cytochrome oxidase exhibits phosphorescence from tryptophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween-20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern-Volmer quenching constant of approximately 5 x 10(7) M-1.s-1 at 5 degrees C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from tryptophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen. 相似文献
9.
Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans. 总被引:2,自引:2,他引:0
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The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed. 相似文献
10.
Moncrieffe MC Juranic N Kemple MD Potter JD Macura S Prendergast FG 《Journal of molecular biology》2000,297(1):147-163
Heterogeneous fluorescence intensity decays of tryptophan in proteins are often rationalized using a model which proposes that different rotameric states of the indole alanyl side-chain are responsible for the observed fluorescence lifetime heterogeneity. We present here the study of a mutant of carp parvalbumin bearing a single tryptophan residue at position 102 (F102W) whose fluorescence intensity decay is heterogeneous and assess the applicability of a rotamer model to describe the fluorescence decay data. We have determined the solution structure of F102W in the calcium ligated state using multi-dimensional nuclear magnetic resonance (NMR) and have used the minimum perturbation mapping technique to explore the possible existence of multiple conformations of the indole moiety of Trp102 of F102W and, for comparison, Trp48 of holo-azurin. The maps for parvalbumin suggest two potential conformations of the indole side-chain. The high energy barrier for rotational isomerization between these conformers implies that interwell rotation would occur on time-scales of milliseconds or greater and suggests a rotamer basis for the heterogeneous fluorescence. However, the absence of alternate Trp102 conformers in the NMR data (to within 3 % of the dominant species) suggests that the heterogeneous fluorescence of Trp102 may arise from mechanisms independent of rotameric states of the Trp side-chain. The map for holo-azurin has only one conformation, and suggests a rotamer model may not be required to explain its heterogeneous fluorescence intensity decay. The backbone and Trp102 side-chain dynamics at 30 degrees C of F102W has been characterized based on an analysis of (15)N NMR relaxation data which we have interpreted using the Lipari-Szabo formalism. High order parameter (S(2)) values were obtained for both the helical and loop regions. Additionally, the S(2) values imply that the calcium binding CD and EF loops are not strictly equivalent. The S(2) value for the indole side-chain of Trp102 obtained from the fluorescence, NMR relaxation and minimum perturbation data are consistent with a Trp moiety whose motion is restricted. 相似文献
11.
Phosphorescence anisotropy from internal tryptophan (Trp) residues in proteins which are in the crystalline state may provide an experimental approach suitable to study the flexibility of rather rigid segments of protein structure. The phosphorescence anisotropy of Trp-314 in liver alcohol dehydrogenase, which is enclosed within the beta-sheet forming the coenzyme-binding domain, was measured with the protein free in solution and in the crystalline state. In contrast to the free protein, where the rotational correlation time reflects the tumbling rate of the whole macromolecule, there is effectively no loss in anisotropy in the crystalline state. At room temperature, the triplet lifetime of 0.5 s implies that the rotational correlation time of the indole side chain must be larger than 1 s. Anisotropy data show that fluctuations of the indole ring about the average position can only be of limited amplitude (cone of semiangle less than 15 degrees) and that the resistance opposed by the beta-sheet to out-of-plane rotational motions is equivalent to a viscosity larger than 2.5 X 10(8) P, a value which confirms the particular rigidity anticipated for such an assembly of secondary structure. 相似文献
12.
The triplet-triplet absorption spectrum of the sole indole side chain of human serum albumin and its decay kinetics were previously characterized, at room temperature, by using a conventional flash photolysis method [(1978) Proc. Natl. Acad. Sci. USA 75, 1172-1175]. Exploitation of this potentially useful long lived reporter group in protein studies was limited by the excessively large sample size required by that apparatus. The 265 nm laser flash instrument used in the present work avoids this problem at the price of a loss in photo-selectivity. We report that the latter concern can be mitigated. Melittin was studied first because this polypeptide contains a single aromatic residue (W-19), and because its monomeric and tetrameric forms are good models for solvent exposed and buried indole side chains of proteins. For both forms, the indole triplet and neutral radical absorption spectra could be readily time resolved and identified on the basis of shape and differential dioxygen sensitivity. The single tryptophan containing protein human serum albumin was studied next because it contains a large number of other 265 nm absorbing moieties whose transient spectra might complicate the detection of the indole triplet. These transients were shown to not interfere significantly in the wavelength region 450 nm to 600 nm, and, in contrast to the indole triplet, they were relatively dioxygen insensitive. Thus, a facile means is available by which the indole triplet of proteins may be characterized. Subsequently the question of whether this species could be detected in the presence of nuclei acid components was investigated by flashing the phage fd. The putative nucleic acid transients were shown not to interfere and the absorbance of the indole triplet was readily time resolved. The spectral assignment was persuasively confirmed by showing that the indole triplet absorption and phosphorescence emission spectra decay with the same lifetime. The present work thus provides additional evidence for the general applicability of the indole triplet excited state as a long lived intrinsic protein reporter group. 相似文献
13.
Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan
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Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes. 相似文献
14.
Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane. 相似文献
15.
A spectroscopic probe of stacking interactions between nucleic acid bases and tryptophan residues of proteins. 总被引:1,自引:0,他引:1
The external heavy atom effect of mercury on the spectroscopic properties of the indole ring has been used to investigate stacking interactions of tryptophan with mercurinucleotides in mixed aggregates formed in frozen aqueous solutions as well as in oligopeptide-polynucleotide complexes. This effect is characterized at 77 K by a quenching of the tryptophan fluorescence, an enhancement of the phosphorescence emission and a drastic shortening of the phosphorescence lifetime. These phenomena result from an enhanced spin-orbit coupling due to a close contact between the mercury atom and the indole ring. Dissociation of the complexes leads to a recovery of the spectroscopic properties of the free tryptophan ring. The possible use of this spin-orbit probe to provide evidence for stacking interactions in protein-nucleic acid complexes is discussed. 相似文献
16.
Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm. 相似文献
17.
D'Auria S Staiano M Varriale A Gonnelli M Marabotti A Rossi M Strambini GB 《Proteins》2008,71(2):743-750
Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP. 相似文献
18.
Energy transfer between excited triplet states of aromatic amino acid residues was observed at 1.4 degrees K. The distance necessary for energy transfer between monomeric tyrosine and tryptophan residues was determined to be roughly 63 A. Total phosphorescence decay rate constants for several proteins were determined while emission corresponding to tyrosine and tryptophan residues was monitored. The observed decay rate constants are interpreted in terms of intramolecular interactions of the polypeptide residues. 相似文献
19.
The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
The decay of Trp phosphorescence of proteins in fluid solutions was shown to provide a sensitive tool for probing the conformational homogeneity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential decays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which the conformer equilibrium is derived, was found to be a sensitive function of solvent composition (buffer, pH, ionic strength and glycerol cosolvent), temperature, and complex formation with substrates and cofactors. In many cases, raising the temperature, a point is reached at which the decay becomes practically monoexponential, meaning that conformer interconversion rates have become commensurate with the triplet lifetime. Estimation of activation free energy barriers to interconversion shows that the large values of DeltaG* are rather similar among polypeptides and that the protein substates involved are sufficiently long-lived to display individual binding/catalytic properties. 相似文献