Ionization Potentials of Fluoroindoles and the Origin of Nonexponential Tryptophan Fluorescence Decay in Proteins ⊥ (original) (raw)
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Journal of the American Chemical Society, 1990
- Pernot, C.; Lindqvist, L. J . Photochem. 1976/1977, 6 , 215-220. (54) Strickland, E. H.; Billups, C.; Kay, E. Biochemistry 1972, 11, 3657-3662.
Chemical Physics, 2008
Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl-and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.
Intramolecular Quenching of Tryptophan Fluorescence by the Peptide Bond in Cyclic Hexapeptides
Journal of the American Chemical Society, 2002
Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide c[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D-and 2D-1 H NMR techniques. The peptide backbone has a single predominant conformation. The tryptophan side chain has three 1 rotamers: a major 1 ) -60°rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the 1 rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the 1 ) -60°rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to peptide bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the 1 ) 180°rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the -ammonium does not occur in these peptides.
Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium
Journal of Physical Chemistry A, 2009
Fluorescein is one of most used fluorescent labels for characterising biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins, then one must account for the fact that the fluorescence of fluorescein labelled protein can be affected by the presence of intrinsic amino acids residues, such as, tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved and this makes it difficult to evaluate quantitatively the photophysics of fluorescein labelled proteins. To address this we have explored the fluorescence of fluorescein in buffered solutions, in different acid and basic conditions, and at varying concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Non-fluorescent complexes with low association constants (5.0-24.1 M-1) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphereof-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3×10 9 M-1 s-1) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer mechanism was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7×10 9 s-1) and the electronic coupling values (5.7-25.1 cm-1) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analysed. This data will be applied to generate a more detailed, quantitative understanding of the photophysics of fluorescein when conjugated to proteins containing the amino acid tryptophan.
Photochemistry and Photobiology, 1985
Abstractxharge effects on the quenching of tryptophan fluorescence in small peptides by iodide ion have been analyzed by the conventional "static" quenching model and by a recently proposed competitive quenching model. The former involves a fit of the quenching data using two quenching parameters-me for dynamic and one for static quenching contributions. The latter model involves a single parameter fit in which the fitting parameter is the characteristic rate constant for quenching of the fluorescent state. Both models indicate a clear charge effect on the efficiency of quenching by iodide ion. Howevcr, the static model results are obscured by the interdependence of the two fitting parameters and the fact that the true physical meaning of the static parameter is uncertain. Rate constants derived from the competitive model can be converted into relative quenching efficiencies. These efficiencies, which vary by more than a factor of two for the molecules studied, are greatest when the positive charge ison the tryptophan and least when this residuc contains a negative charge.
Peptide Sequence and Conformation Strongly Influence Tryptophan Fluorescence
Biophysical Journal, 2008
This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.
Biochemistry, 1992
A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (10.2) and 0.74 (fO.l) ns at pH 8.9, and 0.89 (f0.05) and 0.65 (10.05) ns at pH 5.8. In the mutant Trp-94 -Phe the lifetime of Trp-71 is 4.73 (f0.008) ns at high pH and 4.70 (10.004) ns at low pH. In the mutant Trp-71 -Tyr, the lifetime of Trp-94 is 1.57 (f0.03) ns at high pH and 0.82 (f0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Theor. Chim. Acta 24, 265-2701. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all f2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Forster [ Forster, T. (1948) Ann. Phys. (Leipzig) 2, . The fluorescence lifetime of Trp-94, as determined in a mutant which lacks Trp-7 1, is heavily quenched by the neighboring imidazole group of His-1 8.
Nonexponential fluorescence decay in tryptophan and tryptophan-containing peptides and proteins
NONEXPONENTIAL DECAY IN TRYPTOPHAN The major difficulty in using tryptophan fluorescence decay to probe the structure and dynamics of peptides and proteins is the intrinsic nonexponential decay of tryptophan itself. We have recently proposed a model which rationalizes the nonexponential deca¥ of Trp in terms of conformers about the Ca_CB, or x , bond. See figure 1. The underlying assumption of this
Protein Science, 2008
A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor~NSCP! changes its conformation upon binding of Ca 2ϩ or Mg 2ϩ. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21~W32! and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.
Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence
Proceedings of the National Academy of Sciences, 1990
Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface-i.e., kq = Aexp(-r/p), where A is the effective area of the protein. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electrontransfer rate on distance in a protein environment and evaluate the distance parameter, p, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donoracceptor pair with ket = k~exp(-r/p), ko 109 sec'1.