Proteins, fluorescence globular

B. Somogyi, J. A. Norman, and A. Rosenberg, Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins, Biophys. J. 50, 55-61 (1986). 

Vasilescu, M., Angelescu, D., Almgren, M., Valstar, A. (1999). Interactions of globular proteins with surfactants studied with fluorescence probe methods. Langmuir, 15, 2635-2643. 

Because of the importance of validating the moist-heat treatments and related processes, it is necessary to develop a BI that can efficiently assess the success of the treatment. The use of a fluorescent marker designed for a quick and reliable assay, detectable by microscopy, spectrofluorometry, or a handheld UV lamp, is examined herein. GFPuv provides the basis for its potential utility as a fluorescent BI, to monitor moist-heat treatments (T < 100°C). GFPuv is a compact, globular acidic protein (p/ 4.6-5.4) with one fluorophore consisting of a cyclic tripeptide in the primary protein sequence, a chain of 238 amino acids. It has been shown to be resistant to heat (T > 70°C) and alkaline pH (between 5.5 and 12.0 optimum = 8.0). 

One of the primary mechanisms of protein degradation is the loss of globular structure [118, 119]. This process, termed denaturation, leads to a partially or completely unfolded species which usually lacks any of the biological activity of the native protein. A variety of methods have been employed to monitor the denaturation of proteins, including fluorescence, infrared, nuclear magnetic resonance (NMR), and CD spectroscopy. As CD is very sensitive to changes in both secondary and tertiary structure, its application to the study of protein folding... 

Figure 10.4 (A) A schematic of globular actin monomer forming a protein filament, called F-actin. This filament is one of the important components of muscle cells, as well as the cytoskeleton of other cells. (B) Oriented actin filaments inside a fibroblast cell, called stress fibers, seen through a fluorescence microscope. Image obtained from Nguyen et al. [148] and reprinted with permission.

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. 

The efficiencies of excitation energy transfer from tyrosine to tryptophan residues in globular proteins in native and denatured states has been measured by studying the wavelength dependence of the fluorescence quantum yield.The results are summarized in Table 25. Unlike findings from earlier work, energy transfer is almost completely absent in the denatured state. 

Figure 2.24 Typical fluorescence excitation and emission spectra for a globular protein in aqueous buffer at room temperature, The excitation wavelength, Aexc, is 290 nm arrow). The excitation spectrum baseline measured with buffer in the absence of protein is shown offset tor clarity...

Figure 2.26 Stern-Volmer plots lor quenching of fluorescence in (a) tryptophan in solution (b) tryptophan residues in a globular protein, by quencher molecule, Q. The smaller slope (/

Figure 6.4 Example of stopped-flow fluorescence changes for refolding of a globular protein in solution...

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