Fluorophores protein intrinsic fluorescence

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. 

The three proteins show intrinsic fluorescence due to the presence of Trp residues. Thus, although binding occurs between LCA and LTF or STF, we cannot use fluorescence of Trp residues of LCA to follow this interaction, since there will bean overlapping with the fluorescence of Trp residues of LTF or STF. Therefore, it is necessary to use an extrinsic probe, which is bound to one protein only. A covalently bound fluorophore such as fluorescein is very suitable to perform binding experiments, since there will be no real binding between the fluorophore and the added protein, c Figure 13.6 shows the fluorescence intensity at 515 nm of fluorescein bound to LCA in the presence of increasing concentrations of LTF or STF. 

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. 

When a protein contains two classes of intrinsic fluorophore, one at the surface of the protein and the second embedded in the protein matrix, fluorescence intensity quenching with cesium or iodide allows obtaining the spectra of these two classes. A selective quenching implies that addition of quencher induces a decrease in the fluorescence observables (intensity, anisotropy and lifetime) of the accessible class. At high quencher concentration the remaining observables measured will reflect essentially those of the embedded fluorophore residues. In this case, one can determine the fraction of fluorescence intensity that is accessible (fa). Knowing fa along the emission spectrum will allow us to draw the spectrum of each class of fluorophore (Lehrer, 1971). 

Discussions of biochemical fluorescence h uently start with the subject of wot n fluorescence. Hiis is because, among biopolymers. wotons are unique in dis rf ing useful intrinsic fluorescMice. Lipids, membranes, and saccharides are essenrially nonfluoresc t. and the intrinsic fluorescence of DNA is too weak to be useful In proteins, the three anxnatic amino acids— dienylalanine. t osine. and try ptc han—are all fluorescent A favorable feature of protein structure is that these three amino acids are relatively rare in proteins. Tryptophan, which is the dominant intrinsic fluorophore, is generally present at about 1 mol % in proteins. A protein may possess just one or a few tryptophan residues, which facilitates interpretation of the spectral data. If all 20 amino acids were fluorescent, it is probable that arotein emission would be too complex to int ret. 

Intrinsic fluorophores are naturally occurring whereby the intrinsic fluorescence originates within the aromatic amino acids such as tryptophan, tyrosine, and phenylalanine. The indole groups of tryptophan residues are the dominant source of UV absorbance/emission in proteins. 

Photoluminescence can be used to detect an analyte in three ways (1) the analyte itself is intrinsically fluorescent (direct sensing) (2) the analyte can be tagged with a fluorophore label or (3) the analyte interacts with a luminescent probe. Direct sensing and fluorophore-tags are widely used in biomedical applications to probe cell environments. Many proteins are intrinsic fluorophores due to the presence of the aromatic amino acids tryptophan, phenylalanine and tyrosine. Analytes such as pH, CO2, NH3, O2 and various cations and anions can be measured indirectly using luminescence probes. 

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). 

A fluorophore is a component of a protein or small molecule that exhibits fluorescence. It can also be called a fluorescent label, chromophore, or fluorescent probe. Each fluorophore has characteristic excitation and emission wavelengths. In other words, a fluorophore will fluoresce when the light of a particular energy, corresponding to the excitation wavelength, is used. Examples of fluorophores include GFP and related emissive proteins, and small molecules like fluorescein and coumarin. Many biomolecules have intrinsic fluorescence. For instance, tryptophan, an amino acid, fluoresces in the ultraviolet region of the electromagnetic spectrum. 

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]. 

The fluorescence energy transfer process has been widely used to determine the distance between fluorophores, the surface density of fluorophores in the lipid bilayer, and the orientation of membrane protein or protein segments, often with reference to the membrane surface and protein-protein interactions. Membranes are intrinsically dynamic in nature, so that so far the major applications have been the determination of fixed distances between molecules of interest in the membrane. 

A large number of fluorescence decay measurements have been performed with proteins.127 Studies on the fluorescence decay of tyrosine and tryptophan and their derivatives, and on biologically active peptides containing intrinsic or extrinsic fluorophores have also been carried out and a few illustrative examples will be reviewed here. 

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... 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

Therefore, in static quenching, one observes an intensity decrease only. Binding of TNS to a i-acid glycoprotein induces a decrease in the fluorescence intensity of the protein Trp residues. Fluorescence lifetime of the intrinsic fluorophore is not modified. Variations in Trp residue intensities and lifetimes can be analyzed by plotting intensities and lifetimes in the absence and presence of TNS as a function of TNS concentration (Figure 10.8). It is clear from the figure that the TNS- i -acid glycoprotein interaction is of a static nature. 

The fluorophore should be bound to one of the two proteins only (case of extrinsic fluorophores) or should be part of it (case of intrinsic fluorophores). Also, the binding parameters of the fluorophore-macromolecule complex can be determined by following the fluorescence modification of the fluorophore observables. 

The use of fluorophores intrinsic to the protein allows researchers to probe protein structure and dynamics without incorporating non-native fluorophores that could perturb the native structure of the protein. Proteins usually contain one or more fluorescent amino acids, which makes dynamic studies on the native protein feasible. Often, fluorescent amino acid residues can be introduced by site-directed mutation without altering protein structure significantly. 

As described above, the intrinsic fluorophores that nature provides in proteins generally have rather low absorption coefficients and quantum yields. For many applications, brighter fluorescence probes are needed, and emission in the visible region is desirable. One way to meet these needs is by labeling with a fluorescent dye. A wide range of fluorescence probes is now available for this purpose (see Fig. lb). A summary of the spectroscopic and photophysical properties of many fluorescence probes is available in References 5 and 6. 

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