Fluorescent protein definitions

I ll begin by telling you a bit about fluorescent proteins and how they ve revolutionized experimental biology. A key contributor to this area is Professor Roger Y. Tsien of University of California, San Diego (UCSD) As I m sure you know. Professor Tsien, along with Martin Chalfie and Osamu Shimomura, received the 2008 Nobel Prize in Chemistry for the discovery and development of a fluorescent protein called green fluorescent protein (GFP). You should definitely look up his Nobel Prize lecture and maybe even make an effort to visit his laboratory. 

Let us turn our attention back again to the scheme illustrating various versions of the joint application of fluorescence parameters (Figure 2.1) and consider the possibilities for constructing more general and more definite models of protein dynamics. These models can be suggested and confirmed or rejected by comparing predicted behavior with the results of spectroscopic experiments of different kinds. 

From the mere fact that CF, can be released from the membrane by EDTA treatment and the enzyme stays in solution without detergents, it is apparent that the catalytic sector has minimal, if any, direct interaction with the lipids of the chloroplast membrane. It is a globular protein that is held to the surface of the membrane via interaction with the membrane sector. Recently it was shown that the y subunit is in immediate contact with the membrane sector and the 8 and e subunits may induce proper binding for catalysis [17,18], The enzyme contains a few well-defined sites that were used for localization experiments by the method of fluorescent energy transfer [19,56-61], These studies revealed the position of those sites and helped to localize the various subunits of CF, in space relative to the chloroplast membranes (for a model of CF, see Refs. 61 and 62). These experiments are awaiting analysis of the amino acid sequence of the y subunit that is now under investigation in Herrmann s laboratory [148], Definite structural analysis could be obtained only after good crystals of the enzyme become available. 

A direct demonstration of binding of alcohol to LADH in the absence of coenzyme has been made (253) by utilizing the spectroscopic changes and protein fluorescence quenching that occur when the chromophoric substrate hydroxymethyl ferrocene binds. It was not definitively established, however, that the alcohol binds in the substrate binding pocket. 

At alkaline pH the fluorescence of Class B proteins is found to be that of tyrosinate isi-i ). A report of Vladimirov and Zimina )that the fluorescence of serum and egg albumins at pH 13 is entirely due to tryptophan is probably in error the observed luminescence is most likely that of tyrosinate. Tryptophan and t3n-osinate fluorescence spectra are quite similar and hfetime measurements are sometimes necessary for definite identification. The phosphorescence of Class B proteins at alkaline pH generally has a considerable tr5 tophan component along with a dominant tyrosinate contribution 26,164). Thus, trp- -t T < > transfer appears to be very efficient at the singlet level and enhanced intersystem crossing to the tryptophan triplet at high pH 26) also contributes to the tryptophan fluorescence quenching and to the production of tryptophan phosphorescence of Class B proteins at high pH. It is possible that tyr( ) - -trp triplet transfer also occurs to an extent in some proteins. 

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

A Judicious use of CO sensing fluorescent probes (Section 1.4) may provide more sohd clues on the chemical biology of these CORMs. Indeed, a first test with COP-1 proves that CORM-3 delivers CO to cells in vitro [54]. It is plausible to speculate that, in vitro, a complex of the type [Ru(CO)2(CO(Y))L3] (Y = nucleophile), hke those in Ref. [38], enters the cell, reverts to [Ru(CO)3Ls], and releases CO, and that similar alternatives operate in vivo eventually mediated by plasma proteins. The existing ESI-MS and FTIR evidence presented earlier should not be taken as definitive due to the nonbiological conditions in which it was obtained. 

Vl s (1946) has reported fluorescence in crystals of amino acids and in proteins both in the solid and in solution. Examination of his data show that the results obtained with amino acids must be due to impurities. Thus, in the case of tyrosine, excitation with the wavelength of nearly maximum absorption resulted in no fluorescence being emitted, while emission took place on excitation with shorter or longer wavelengths. This is exactly what one would consider necessary to show that the fluorescence was due to an impurity and not to the tyrosine itself. A similar criticism applies to V15s results with serum albumin. His conclusion that the results obtained with proteins are explainable on the basis of an antistokes effect rests on a definition of the term which is peculiar to this author. 

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