Intrinsic fluorescence, protein

We have consistently talked about the polarization of a fluor bound to a macromoleeule. What about the intrinsic fluorescence of the macromolecules and its polarization For example the intrinsic fluorescence of tryptophan in a protein and utilization of its polarization in stud3dng the protein. There is a problem here. Large proteins move very slowly on a molecular scale. Thus, to observe depolarization due to motion, the lifetime of the excited state should be sufficiently long, i.e., there should be a good time lag between excitation and emission so that the molecule may show substantial movement in that time and depolarization may occur. For very small proteins, intrinsic fluorescence may be of some use, but for larger proteins, extrinsic fluorescence has to be made use of. 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

The aim of this Chapter is to review a method by which fluorescence properties of organic dyes can, in general, be predicted and understood at a microscopic (nm scale) by interfacing quantum methods with classical molecular dynamics (MD) methods. Some review of our extensive applications [1] of this method to the widely exploited intrinsic fluorescence probe in proteins, the amino acid tryptophan (Trp) will be followed by a discussion of electrochromic membrane voltagesensing dyes. 

Dixit, R., Cyr, R. and Gilroy, S. (2006). Using intrinsically fluorescent proteins for plant cell imaging. Plant J. 45, 599-615. 

L. Zheng and J.D. Brennan, Measurement of intrinsic fluorescence to probe the conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in a sol-gel derived glass matrix. Analyst 123, 1735-1744 (1998). 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

The absorption of the tyrosines in pig intestinal Ca2+-binding protein is reported to be shifted to longer wavelengths the intrinsic fluorescence, however, is in the normal energy region for tyrosine emission with a possibility of some emission from tyrosinate/143, 45) These results can be equally well explained by a ground-state, hydrogen-bonded complex. 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

As shown above, the intrinsic fluorescence spectra of proteins as well as coenzyme groups and probes shift within very wide ranges depending on their environment. Since the main contribution to spectral shifts is from relaxational properties of the environment, the analysis of relaxation is the necessary first step in establishing correlations of protein structure with fluorescence spectra. Furthermore, the study of relaxation dynamics is a very important approach to the analysis of the fluctuation rates of the electrostatic field in proteins, which is of importance for the understanding of biocatalytic processes and charge transport. Here we will discuss briefly the most illustrative results obtained by the methods of molecular relaxation spectroscopy. 

K. K. Turoverov and I. M. Kuznetsova, Polarization of intrinsic fluorescence of proteins. 2. The studies of intramolecular dynamics of tryptophan residues, Mol. Biol. (Moscow) 17, 468-475 (1983). 

B. Somogyi, J. A. Norman, and A. Rosenberg, Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins, Biophys. J. 50, 55-61 (1986). 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

Examples of its use include protein and nucleic acid detection, enzyme-labelled fluorescence, in the intrinsic fluorescence of normal and cancer cells, as external... 

The determination of fluorescence parameters of peptides requires the presence of either natural fluorescent amino acid residues (intrinsic fluorescence) or of extrinsic fluorescent probes covalently attached to the peptide at appropriate sites. The use of extrinsic fluorescent probes is mandatory in cases where the conformational or rotational behavior of a peptide is examined in the presence of proteins that contain intrinsic fluorescent amino acids. 

Most interesting applications of intramolecular energy transfer between nonconjugated chromophores are found in the conformational studies of biomolecules like nucleic acids and proteins. The experiments on rotational depolarization of emission from intrinsic fluorescent groups on externally attached fluorescent probes, have resulted in a vast store of knowledge which has helped to enrich the subject of photobiology. 

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