Fluorescent proteins chromophore formation

Reid, B. G., and Flynn, G. C. (1997). Chromophore formation in green fluorescent protein. Biochemistry 36 6786-6791. 

Sniegowski JA, Lappe JW, Patel HN, Huffman HA, Wachter RM (2005) Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein. J Biol Chem 280 26248-26255... 

Verkhusha W, Chudakov DM, Gurskaya NG, Lukyanov S, Lukyanov KA (2004) Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chem Biol 11 845-854... 

Scheme 5.1 Chromophore formation in green fluorescent protein.

The sensitivity of any reporter system based on fluorescent proteins is determined by numerous factors such as the total amount of fluorescent protein produced in the system, the efficiency of protein maturation/chromophore formation, the individual properties of the fluorescent protein in use, the organism and tissue in which the fluorescent protein is expressed and, last but not least, the available technical equipment [51]. 

The efficiency of protein/chromophore maturation is an intrinsic property of each fluorescent protein or mutant thereof. With respect to this, time, temperature, oxygen-availability and the intrinsic rates of cyclization/oxidation during chromophore formation play important roles [51]. As outlined in this review the latter is strictly dependent on the specific interaction between the chromophore residues and the environmental amino-acid side-chains provided by the 6-can protein backbone. Availability of chaperonins can be helpful [105] but is not required. In case of fusions between host proteins and a fluorescent protein hindrance of the protein folding thus preventing proper maturation can not be excluded. This can only by tested empirically. 

Branchini, B.B., Nemser, A.R., and Zimmer, M., A computational analysis of the unique protein induced tight turn that results in posttranslational chromophore formation in green fluorescent protein, /. Am. Chem. Soc., 120, 1-6,1998. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Even lower temperatures have been used to study possible intermediate stages in the formation of the acyl enzyme. A tetrahedral intermediate (with a covalent bond between the substrate carbonyl carbon atom and the oxygen atom of the active site serine) (Fig. 2) had been suggested by analogy with nonenzymatic reactions. With rapid reaction techniques, spectrophotometric evidence has been obtained for an additional intermediate before the acyl enzyme in the case of chromophoric substrates. By using first the protein fluorescence emission (Fink and Wildi, 1974)... 

The most critical and demanding requirement is that the fluorescent product generated by an enzyme is retained quantitatively within the cell. A number of ingenious strategies for product retention have been developed including intracellular precipitation (the Enzyme-Linked Fluorescence or ELF family of fluorescent substrates), attachment of lipophilic anchors, substrates that yield insoluble chromophores, and retention via formation of complexes with intracellular proteins (for examples, see footnote 3 and Zlokamik et al., 1998). 

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