Native fluorescent proteins

In addition to native fluorescence, proteins may be labeled with three different types of fluorescent labels. The most important examples are given in Table 8 and the formulae of some of them are shown below (Fig. 7). 

In the past several years, our group has adapted genes that encode fluorescent proteins for the study of several aspects of mRNA polyadenylation in plants. In particular, we have used native fluorescent protein reporters as well as proteins containing a nuclear localization signal to smdy the properties of poly(A) sites and signals as well as of proteins that are part of the polyadenylation complex. These various genes have been used in conjunction with J robacterium-mcdia.ted transient infection to rapidly assess different parameters. In this report, these methods are... 

There has been interest in miniaturizing and automating electrophoresis of proteins. Ruchel (1997) reported a miniaturized system where proteins are first separated by isoelectric focusing in millimeter diameter tubes. The tube s contents are transferred to a slab gel that is a few centimeters on a side. This technology was used to separate the proteins from a single giant neuron from Aplasia califomicus. More recently, native fluorescence has been used to resolve 200 proteins from a similar miniaturized electrophoresis system (Sluszny and Yeung, 2004). 

Sluszny, C., Yeung, E.S. (2004). One-and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal. Chem. 76, 1359-1365. 

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fluorescence detection offers the possibility of high sensitivity and, in the case of complex samples, improved selectivity. However, this mode of detection requires that the analyte exhibit native fluorescence or contain a group to which a fluorophore can be attached by chemical derivatization. Because only tryptophan and tyrosine exhibit significant native fluorescence, fluorescence detection of proteins usually requires derivatization. 

DOTMA E. coli E EBV ECFP ECV EGFP ELISA EYFP FACS FdG FH2 FH4 FK506 FLP propane-aminium-trifluoracetate 7V-[2,3-(dioleyloxy) propyl]-/V,/V,/V-trimethyl ammonium chloride Escherichia coli erythromycine operon/repressor Epstein-Barr virus enhanced cyan fluorescence protein extracellular viral particles enhanced green fluorescence protein enzyme-linked immunosorbent assay enhanced yellow fluorescence protein fluorescence-activated cell sorter fluorescein di- 3-D-galactopyranoside dihydrofolate tetrahydrofolate human immunophilins native recombinase isolated from the 2pm plasmid from Saccharomyces cerevisiae... 

The riboflavin binding protein that occurs in eggs has been exploited for the radio-ligand binding assay of riboflavin. Because binding to the protein quenches the native fluorescence of riboflavin, it can be exploited for a direct titrimetric fluorescence assay of the vitamin in urine and other biological samples (Kodentsova et al., 1995). 

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. 

The three aromatic amino acids, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), are the only native amino acids with useful fluorescence properties. Figure la shows their absorption and fluorescence spectra. Their fluorescence properties are summarized in Table 1. Note that the relative absorption coefficients increase in the order Phe < Tyr < Trp. The fluorescence quantum yields increase in the same order. The product of absorption coefficient and fluorescence quantum yield can be taken as a measure of the brightness of the fluorophore. By the standards of fluorescent dyes (see below), the brightness of aU three amino acids is poor. Trp is the brightest of the three, and for proteins with a small number of Trp residues it may be possible to assign fluorescence decays to specific Trp residues. As a result, of the three fluorescent amino acids, Trp is by far the most widely exploited for its fluorescent properties. Fluorescence from Tyr is also detectable but may be masked by Trp fluorescence. Proteins often contain many Tyr residues, so it is often not possible to isolate the fluorescence from individual Tyr residues. Fluorescence from Phe is weak and not often used in fluorescence studies. 

The fluorescence intensity resolved by wavelength constitutes the fluorescence spectrum. The wavelengths of fluorescence photons contain information about the environment of the fluorophore and the sample heterogeneity. Por example, as described above, buried Trp residues tend to have blue-shifted emission bands (Xmax < 330 mn), whereas Trp residues partially or fully exposed to water have red-shifted emission bands ( max > 340 mn). Therefore, protein conformational changes or unfolding may be accompanied by shifts in the native fluorescence spectra. Pluorescence spectra can be measured on a standard fluorometer, which is available from many manufacturers. 

For those without native fluorescence, two common approaches have been employed, namely, derivatization and indirect fluorescence. Derivatizing agents should be pure, low fluorescent, and stable, as well as react quickly and uniquely with the analytes and, thus, formed compounds should be strongly fluorescent and stable. These include dansyl chloride, fluorescamine, 4-clair-7-nitro-benz-2-oxa-l,3-diazole (NBD), o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA), which have been used for the analyses of amino acids, peptides, proteins, thiols, and sugars with LOD in 1-100 nM range. Compared to LINF, approaches based on derivatization provide the advantages of relatively low cost and versatility in instrumentation, but they may suffer from contamination and loss of temporal information. 

Laser-induced native fluorescence is measured on a set of bio-molecules from different classes (bacteria, proteins, fungi) for excitation at 266nm and 355nm. A method of preprocessing the spectra to obtain an inherently normalized set of data is described. Class identification on the normalized data set is demonstrated. 

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