Protein extrinsic fluorescence

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. 

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)... 

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. 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

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. 

In other media like micelles, cyclodextrin, binary solvent mixtures, and proteins (47-55), lifetime distributions are routinely used to model the decay kinetics. In all of these cases the distribution is a result of the (intrinsic or extrinsic) fluorescent probe distributing simultaneously in an ensemble of different local environments. For example, in the case of the cyclodextrin work from our laboratory (53-55), the observed lifetime distribution is a result of an ensemble of 1 1 inclusion complexes forming and coexisting. These complexes are such that the fluorescent probe is located simultaneously in an array of environments (polarities, etc.) in, near, and within the cyclodextrin cavity, which manifest themselves in a distribution of excited-state lifetimes (53-55). In the present study our experimental results argue for a unimodal lifetime distribution for PRODAN in pure CF3H. The question then becomes, how can a lifetime distribution be manifest in a pure solvent ... 

Fluorescence measurements on proteins require both an appropriate fluorescence technique and the presence of a suitable fluorophore. The techniques used for the application of fluorescence to proteins are described later in this article. In this section, we briefly consider three classes of fluorophores that are used widely to study proteins native fluorophores including fluorescent amino acids, extrinsic fluorescent labels, and auto-fluorescent proteins. Each has advantages for probing proteins and has distinct drawbacks No perfect fluorophore exists for studying proteins. 

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

Fluorescein isothiocyanate (FITC) and dansyl-chloride were among the first extrinsic fluorescent labels for proteins used for immunofluorescence microscopy and polarization measurements. Fluorescein and rho-damine labels were extensively employed due to their bright emission in the visible range. These probes have drawbacks including hydrophobicity, small Stokes shifts, and sensitivity to pH and photobleaching, which led to the development of new dyes such as Alexa, Bodipy, and cyanine dye families (see Table 1). These dyes cover a broad... 

In addition to the complicated response of the fluorophore to various stimuli, one more aspect should be home in mind. Only a small number of systems contain intrinsic fluorophores and are inherently fluorescent. Such systems (e.g., tryptophan-containing proteins) can be studied directly and reliable information on the positions, mobility, and accessibility of tryptophan residues for different molecules can be relatively easily obtained. In a majority of cases, a successful fluorescence study requires the addition of a low content of an extrinsic fluorescent probe, which modifies not only optical but also other properties of the studied system. An extrinsic probe feels only the effect of its immediate microenvironment, which has undoubtedly been altered by its insertion. Even though the change in the system is negligible at a macroscopic level, most fluorescence methods report the behavior of the tiny perturbed part of the system. Therefore, the extent and nature of possible perturbation of the system must also be investigated to enable description of the behavior of the unperturbed system. 

Hawe A, Sutler M, Jiskoot W (2008) Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res 25 1487-1499... 

A variety of extrinsic fluorophores can be attached to proteins to serve as fluorescence probes. These can be selected to maximize sensitivity and to avoid contamination (i.e., by moving to longer absorption and emission wavelengths) from other absorbing components. With both intrinsic and extrinsic fluorescence probes, the method focuses only on these probes sites, which might be as few as a single site on a protein. 

Fluorescence anisotropy values for the fiuorescence of a fluorophore on a protein will depend on the fluorophore s rotational freedom and fiuorescence lifetime. Because the motional freedom of intrinsic or extrinsic fluorophores will usually increase when a protein unfolds, a change in a protein s fluorescence anisotropy is expected upon unfolding. However, to properly use anisotropy to analyze the thermodynamics (or kinetics) of an unfolding transition, Eq. (1) should be replaced with one that includes the fluorescence quantum yield of the protein s structural states (see Reference 19). 

The use of an extrinsic fluorescence probe bound to a protein is not recommended for the study of the folding process since the presence of the probe may modify the pathway of folding. 

The conformation of bovine myelin basic protein (MBP) in AOT/isooctane/water reversed micellar systems was studied by Waks et al. 67). This MBP is an extrinsic water soluble protein which attains an extended conformation in aqueous solution 68 but is more density packed at the membrane surface. The solubilization of MBP in the AOT reversed micelles depends on the water/AOT-ratio w0 68). The maximum of solubilization was observed at a w0-value as low as 5.56. The same value was obtained for another major protein component of myelin, the Folch-Pi proteolipid 69). According to fluorescence emission spectra of MBP, accessibility of the single tryptophane residue seems to be decreased in AOT reversed micelles. From CD-spectra one can conclude that there is a higher conformational rigidity in reversed micelles and a more ordered aqueous environment. 

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. 

The fast, sensitive, reliable, and reproducible detection of (bio)molecules including quantification as well as biomolecule localization, the measurement of their interplay with one another or with other species, and the assessment of biomolecule function in bioassays as well as in vitro and in vivo plays an ever increasing role in the life sciences. The vast majority of applications exploit extrinsic fluorophores like organic dyes, fluorescent proteins, and also increasingly QDs, as the number of bright intrinsic fluorophores emitting in the visible and NIR is limited. In the near future, the use of fluorophore-doped nanoparticles is also expected to constantly increase, with their applicability in vivo being closely linked to the intensively discussed issue of size-related nanotoxicity [88]. 

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... 

Quenching of fluorescence of tryptophan residues, coenzyme fluoro-phores, or extrinsic probes buried in the interior of proteins by colli-sional quencher molecules diffusing through the protein matrix/7,25 27)... 

If the soluble protein that specifically adsorbs to the fiber can be extrinsi-cally labeled, the background problem can be avoided. Of course, in vivo proteins cannot be labeled. However, it is conceivable that a protein labeled with a bulky extrinsic group (e.g., fluorescent dextrans) could be confined by a molecular sieve membrane (e.g., a dialysis membrane) within a closed volume surrounding the specifically derivatized optical fiber. When exposed to the (unlabeled) protein in the biological fluid under investigation, the membrane-clad fiber would allow some unlabeled protein to permeate in and... 

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