Quenching of room temperature protein phosphorescence by added small molecules (original) (raw)
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Protein tryptophan accessibility studied by fluorescence quenching
Biochemistry and Molecular Biology Education, 2002
A laboratory class is described to introduce the biochemistry major students to the basic concepts and various applications of fluorescence spectroscopy. Through simple and inexpensive experiments the students learn how to record excitation and emission spectra and measure intrinsic protein fluorescence and its quenching to elucidate the local tryptophan environment. Free tryptophan, ovalbumin, and bovine serum albumin are used for the experiments. The structural information that can be retrieved from emission spectra, as well as from quenching data, is discussed.
Protein fluorescence quenching by small molecules: Protein penetration versus solvent exposure
Proteins: Structure, Function, and Genetics, 1986
Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion. Abbreviations used: AP, alkaline phosphatase; DEAE, diethyaminoethyl; EDTA, ethylene diamine tetraacetate; HSA, human serum albumin; LADH, horse liver alcohol dehydrogenase;
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.
Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins
European Journal of Biochemistry, 1992
The dependence of the fluorescence emission maximum of the tryptophan residues in several twotryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A,) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case ofmetalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.
Biophysical Journal, 1994
The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH), Trp-314, and of alkaline phosphatase (AP), Trp-1 09, show nonexponential phosphorescence decays when the data are collected to a high degree of preciskin. 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-normnalized width of the MEM distributn 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 funrdnentat, 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 byptophan 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 kxca/ rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers.
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.
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.
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.
DISTANCE-DEPENDENT FLUORESCENCE QUENCHING OF TRYPTOPHAN BY ACRYLAMIDE
Photochemistry and Photobiology, 1994
We report results of frequency-domain and steady-state measurements of the fluorescence quenching of p-bis-[2-(5-phenyloxazolyl)]benzene (POPOP) when quenched by bromoform (CHBr 3 ), methyl iodide (CH 3 I), potassium iodide (KI), 1,2,4-trimethoxybenzene (TMB), or N,N-diethylaniline (DEA). The quenching efficiency of these compounds decreased in the order DEA, TMB, KI, CH 3 I, CHBr 3 . In the case of DEA and TMB the measurements clearly confirm the applicability of the exponential distance-dependent quenching (DDQ) model, in which the bimolecular quenching rate k(r) depends exponentially on the fluorophore-quencher separation r, k(r) ) k a exp[-(ra)/r e ], where a is the distance of closest approach. Simultaneous analysis of the frequency-domain and steady-state data significantly improved resolution of the recovered molecular parameters k a and r e . The data for DEA and TMB cannot be satisfactorily fit using either the Smoluchowski or Collins-Kimball radiation boundary condition (RBC) model. The quenching behavior of the less efficient quenchers KI, CH 3 I, and CHBr 3 can be adequately described with both the DDQ and RBC models, but this may be a simple consequence of less efficient quenching. The efficiency of quenching is discussed on the basis of the mechanisms of interaction between the fluorophore and quencher molecules, which involves electron transfer and/or heavy atom effects.