Proteins, fluorescence structure

Determination of protein secondary structure has long been a major application of optical spectroscopic studies of biopolymers (Fasman, 1996 Havel, 1996 Mantsch and Chapman, 1996). These efforts have primarily sought to determine the average fractional amount of overall secondary structure, typically represented as helix and sheet contributions, which comprise the extended, coherent structural elements in well-structured proteins. In some cases further interpretations in terms of turns and specific helix and sheet segment types have developed. Only more limited applications of optical spectra to determination of tertiary structure have appeared, and these normally have used fluorescence or near-UV electronic circular dichroism (ECD) of aromatic residues to sense a change in the fold (Haas, 1995 Woody and Dunker, 1996). 

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

J. R. Lakowicz and G. Weber, Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale, Biochemistry 12, 4171-4179 (1973). 

The properties of membranes commonly studied by fluorescence techniques include motional, structural, and organizational aspects. Motional aspects include the rate of motion of fatty acyl chains, the head-group region of the phospholipids, and other lipid components and membrane proteins. The structural aspects of membranes would cover the orientational aspects of the lipid components. Organizational aspects include the distribution of lipids both laterally, in the plane of the membrane (e.g., phase separations), and across the membrane bilayer (phospholipid asymmetry) and distances from the surface or depth in the bilayer. Finally, there are properties of membranes pertaining to the surface such as the surface charge and dielectric properties. Fluorescence techniques have been widely used in the studies of membranes mainly since the time scale of the fluorescence lifetime coincides with the time scale of interest for lipid motion and since there are a wide number of fluorescence probes available which can be used to yield very specific information on membrane properties. 

Tryptophan fluorescence is very sensitive to the local environment. In an environment with a low polarity, tryptophan emits at a maximum of 320 nm. The peak position shifts to 355 nm in the presence of a polar environment. The loss of the protein tertiary structure (complete denaturation) induces a shift in tryptophan fluorescence to 355 nm. 

Tyrosine is more fluorescent than tryptophan in solution, but when present in proteins, its fluorescence is weaker. This can be explained by the fact that the protein tertiary structure inhibits tryosine fluorescence. Also, energy transfer from tyrosines to tryptophan residues occurs in proteins inducing a total or important quenching of tyrosine fluorescence. This tyrosine — tryptophan energy transfer can be evidenced by nitration of tyrosine residues with tetranitromethane (TNM), a highly potent pulmonary carcinogen. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on the phosphorylation and dephosphorylation of tyrosine, and the subsequent effects on cell proliferation, can be investigated. 

Fluorescence spectra of the tryptophanyl residues of the fully denatured protein is normalized, as is its absorption spectra. In the absence of any structured protein, fluorescence of tryptophanyl residues and of free Trp in solution can be compared. 

When a protein possesses two or several Trp residues, when quenchers such as iodide, cesium, or acrylamide are used, and if all Trp residues are not accessible to the quencher, the Stern-Volmer equation yields a downward curvature. In this case, we have selective quenching (Figure 10.5b). From the linear part of the plot, we can calculate the value of the Stern-Volmer constant corresponding to the interaction between the quencher and accessible Trp residues. Upon complete denaturation and loss of the tertiary structure of a protein, all Trp residues will be accessible to the quencher. In this case, the Stern-Volmer plot will show an upward curvature. In summary, inhibition of the protein fluorescence with two or several Trp residues can yield three different representations for the Stern-Volmer equations, depending on the accessibility of the fluorophore to the quencher. 

R7. Fluorescence of tyrosine in protein is generally quenched as a result of the tertiary structure of the protein. A structural modification of the protein would affect the fluorescence properties of the protein, including those of the tyrosine. Thus, in some cases, one can observe an increase in the fluorescence intensity at 303 nm and the quantum yield of the tyrosine and a decrease in its anisotropy. 

Engelborghs Y. Correlating protein structure and protein fluorescence. J. Fluoresc. 2003 13 9-16. 

Modeling of Multisite Fluorescence Quenching. A traditional multisite fluorescence quenching model using a Stem-Volmer approach has been developed and applied to quenching curves involving residual protein fluorescence (32). More recently, however, the multiple site model has been used to describe structural characteristics in diverse polymer environments (33-3f). The multisite Stem-Volmer model shown in equation 13 may be used to define multiple fluorescent binding sites present under one emission peak... 

Albumin ovalbumin carbonic anhydrase hemoglobin hexokinase MWCNTs MWCNT-COOH MWCNT-tyrosine MWCNT-isobutane amine CD Fluorescence The functionalized MWCNTs selectively induced protein secondary structure changes. Structural changes depend on the enzyme used and the functional group and the concentration of MWCNTs. [136]... 

Barbara Campanini graduated cum laude in pharmaceutical chemistry and technologies, University of Parma in 1998. In 2002, she received her Ph.D. in molecular biology and pathology, University of Parma defending a dissertation on Structural determinants of the stability of the pyridoxal 5 -phosphate-dependent enzyme 0-acetylserine sulfhydrylase. Since 2006, she works as a research scientist at the University of Parma. Her research interests include the functional characterization of PLP-dependent enzymes involved in cysteine metabolism and the preparation of variants of the green fluorescent protein for structural and spectroscopic studies. 

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