共查询到20条相似文献,搜索用时 31 毫秒
1.
The time-resolved fluorescence emission characteristics of the single tryptophan residue (Trp-59) of horse heart apocytochrome c--the precursor of the intramitochondrial cytochrome c--were studied in aqueous solution. The total fluorescence intensity decay measured over the whole emission spectrum was analyzed as a sum of three or four exponentials by the nonlinear least-squares method, the last model always providing a slight but significant decrease in the chi 2 values. Maximum entropy analysis, recently developed for time-resolved fluorometry (Livesey et al., 1987; Livesey & Brochon, 1987), strongly suggests the existence of a distribution including at least four separate classes of lifetimes. The center values were around 0.1-0.2, 1, 3, and 5 ns, in agreement with the lifetime values obtained by nonlinear least-squares regression analysis. As a function of the emission wavelength, these values remained constant within the experimental error, whereas a redistribution of the fractional amplitudes was observed: the contributions of the short components increased in the blue edge region of the emission spectrum. Temperature increase led essentially to a redistribution of the fractional amplitudes, affecting mostly that of the 5-ns component, which almost totally disappeared at high temperature (35-40 degrees C). The lifetime values were not significantly affected except for the 3-ns component, which decreased by about 15% in the temperature range studied. Such observations strongly suggest that the protein exists under different conformational substates in thermal equilibrium. Time-resolved fluorescence anisotropy measurements evidenced the existence of fast internal rotation of the Trp residue. An average maximum restricted angle of rotation of around 55 degrees was calculated. A second internal motion, slower by 1 order of magnitude, corresponding likely to a local motion of the peptide chain involving the Trp-59 residue, was detected on the anisotropy decay curve. Finally, the longest correlation time (5 ns) should correspond to the average rotation of the overall protein. Its value doubled as a function of the protein concentration, revealing an association process leading most likely to a dimer in the concentration range studied (2-139 microM). The flexibility of the peptide chain was more restrained in the associated than in the monomeric form, but the fast internal rotation of the Trp residue was not. 相似文献
2.
Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups. 相似文献
3.
We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein. 相似文献
4.
Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay. 相似文献
5.
A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion. 相似文献
6.
K. Peng A. J. W. G. Visser A. van Hoek C. J. A. M. Wolfs M. A. Hemminga 《European biophysics journal : EBJ》1990,18(5):285-293
The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5. 相似文献
7.
An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor. 相似文献
8.
Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1–1: Microconformational changes induced by S-hexyl glutathione
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Regina W. Wang Andrew W. Bird Deborah J. Newton Anthony Y. H. Lu William M. Atkins 《Protein science : a publication of the Protein Society》1993,2(12):2085-2094
The glutathione S-transferase (GST) isoenzyme A1–1 from rat contains a single tryptophan, Trp 21, which is expected to lie within α-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1–1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9–16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the “H-site” is occupied. 相似文献
9.
We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility. 相似文献
10.
The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1994,1204(2):225-234
The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV vircular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure. 相似文献
12.
Klaus Döring Lars Konermann Thomas Surrey Fritz Jähnig 《European biophysics journal : EBJ》1995,23(6):423-432
The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease TI, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease TI, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypetide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.Abbreviations OmpA
outer membrane protein A
- LP
lactose permease
- RSA
rat serum albumin
- RNAse TI
ribonuclease TI
- P21
21-residue peptide
- NATA
N-acetyltryptophanamide
- PTP
paraterphenyl
- POPE
palmitoyloleoylphosphatidylethanolamine
- POPC
palmitoyloleoylphosphatidylcholine
- POPG
palmitoyloleoylphosphatidylglycerol
- GdHCI
guanidinium hydrochloride
Correspondence to: F. Jähnig 相似文献
13.
14.
Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores. 总被引:1,自引:1,他引:0
The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light. 相似文献
15.
Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein. 相似文献
16.
The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7±0.1 ns and 3.1±0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3±0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of wobbling motion is 35±10° in the tetramer 相似文献
17.
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
18.
K. O. Greulich R. W. Wijnaendts van Resandt G. G. Kneale 《European biophysics journal : EBJ》1985,11(3):195-201
The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs+ than by I-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge.In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried.Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time () of 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a of 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale. 相似文献
19.
Martin Lignell 《Biophysical journal》2010,98(3):425-433
We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the helix-loop-helix scaffold. 相似文献
20.
The evolution of the nanosecond dynamics of the core tryptophan, Trp53, of barstar has been monitored during the induction of collapse and structure formation in the denatured D form at pH 12, by addition of increasing concentrations of the stabilizing salt Na(2)SO(4). Time-resolved fluorescence methods have been used to monitor the dynamics of Trp53 in the intermediates that are populated during the salt-induced transition of the D form to the molten globule B form. The D form approximates a random coil and displays two rotational correlation times. A long rotational correlation time of 2.54 ns originates from segmental mobility, and a short correlation time of 0.26 ns originates from independent motion of the tryptophan side chain. Upon addition of approximately 0.1 M Na(2)SO(4), the long rotational correlation time increases to approximately 6.4 ns, as the chain collapses and the segmental motions merge to reflect the global tumbling motion of a pre-molten globule P form. The P form exists as an expanded form with approximately 30% greater volume than the native (N) state. The persistence of an approximately 50% contribution to anisotropy decay by the short rotational correlation time suggests that the core of the P form is highly molten and permits free rotation of the Trp side chain. With increasing salt concentrations, tight core packing is achieved before secondary and tertiary structure formation is complete, an observation which agrees well with earlier kinetic folding studies. Thus, the equilibrium model developed here for describing the formation of structure during folding faithfully captures snapshots of transient kinetic intermediates observed on the folding pathway of barstar. A comparison of the refolding kinetics at pH 7, when refolding is initiated from the D, P, and B forms, suggests that formation of a collapsed state with a rigid core and approximately 30% secondary and tertiary structure, which presumably defines a coarse native-like topology, constitutes the intrinsic barrier in the folding of barstar. 相似文献