Intrinsic fluorescence probes

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. 

Intrinsic fluorescence probe with the highest fluorescence quantum yield in proteins, which can be exclusively excited at of 290 nm... 

Figure 11.7 Explanatory diagram showing the use ofTryptophan as an intrinsic fluorescence probe for proteins.

Biological Systems - The sensitivity of luminescence techniques and the use of extrinsic and intrinsic fluorescence probes have found considerable applications in biological research. Only a selection of such studies have been selected for citation. 

Another aspect, also considered in Subsection 1.8.3.3.2, concerns fundamental time-resolved fluorescence studies. Here, the emphasis is placed on fiuores-cence depolarization measurements, which are very helpful in following rotational and segmental motions and for studying the flexibility of macromolecules. If the polymer under investigation does not contain intrinsically fluorescent probes (e.g., certain amino acid moieties in proteins), then the macromolecules have to be labeled with fluorescent markers. Information concerning the rate of rotation or segmental motion then becomes available, provided that the emission rate is on a similar time scale. Only when this condition is met can the rate of depolarization be measured. If the emission rate is much faster, there is no depolarization, whereas if it is much slower, the depolarization will be total. 

Small G-proteins contain two regions named switch I and switch II, which undergo a binary conformational change upon nucleotide exchange and GTP hydrolysis (Vetter and Wittinghofer, 2001). The switch II region of Arf and Sar contains a conserved tryptophan residue, which acts as an intrinsic fluorescent probe of the protein conformation. The intrinsic fluorescence of Arfl and Sari increases (by +100% and +200%, respectively) when GDP is replaced by GTP. Tryptophan fluorescence is thus a convenient way to follow the activation-inactivation cycle of these small G-proteins in real-time (Antonny et al, 1997,2001 Bigay et al., 2003 Futai et al, 2004). 

All of these alterations, which may be induced when a protein molecule interacts with a membrane surface either during convective (filtration) processes or under diffusive conditions, can be reported by intrinsic fluorescence probes such as tryptophan residues and detected by using appropriate fluorescence techniques. In fact, the use of these techniques and the correct interpretation of their response is only possible when the number of tryptophan residues present in the protein is relatively low (one or two) because, otherwise, it becomes extremely difficult to assign a given fluorescence response to the corresponding tryptophan, limiting the interpretation effort. 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

We can just measure the intrinsic fluorescence of the target analyte or design sensors based on the variation of the fluorescence of an indicator dye, with the determinand concentration. In the latter case, the probe molecule... 

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). 

Fluorescent probes can be divided into three classes (i) intrinsic probes-, (ii) extrinsic covalently bound probes and (iii) extrinsic associating probes. Intrinsic probes are ideal but there are only a few examples (e.g. tryptophan in proteins). The advantage of covalently bound probes over the extrinsic associating probes is that the location of the former is known. There are various examples of probes covalently... 

C. M. L. Hutnik, J. P. MacManus, D. Banville, and A. G. Szabo, Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102, J. Biol. Chem. 265, 11456-11464 (1990). 

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

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. 

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. 

A suitable fiuorescent probe is an organic molecule, which must change its characteristic parameters with changes in its microenvironment and the parameter must be measurable when the probe is added to the system [54]. The fluorescent probes are categorized as either extrinsic, intrinsic, or covalently bound probes. The intrinsic probes allow a system to be observed without any chemical perturbation. This occurs when the system to be characterized has an in-built fluorescent chromophore unit like tryptophan, tyrosine and phenyl alanine in protein. In some cases the fluorophore is covalently... 

In studies where either intrinsic fluorescence or quenching is a confounding variable, the botanical product should be examined in an assay using a chromatographic separation step with a representative probe substance for the isozyme (29) being examined. 

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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