Fluorescence protein structure analysis using

DT Blankenship, MA Krivanek, BL Ackerman, AD Cardin. High-sensitivity amino acid analysis by derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection applications in protein structure determination. Anal Biochem 178 227-232, 1989. 

Amino acids are derivatized two ways to increase sensitivity. Free amino acids in solution are reacted with o-phthaldehyde (OPA) to form a fluorescent derivative that excites at UV,230nm, and emits at FL, 418 nm. These OPA derivatives are separated on Ci8 in a complex mixture of An/MeOU/ DMSO/water at pH 2.65. PTH amino acids are formed from the N-terminai end of peptides during Edman degradation for structure analysis of peptides and proteins. HPLC is used to identify which amino acids are released. PTH amino acids are separated at UV, 254 nm, on a Ci8 column with a gradient from 10% THF/water containing 5 mM acetic acid to 10% THF/AN.The separation with reequilibration takes 60min. Work with short 3-pm columns has reduced this separation to a 10-min gradient. 

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. 

CSLM can provide focused images to a depth of up to several hundred micrometers, depending on the nature of the sample, so that sequential sections may be obtained for three-dimensional reconstruction of the image (Figure 2). In addition, several chemical components (e.g., protein and fat in cheese) can be identified and localized simultaneously using specific fluorescent labels. CSLM has been used for the quantitative analysis of cellular structures in plant material, the structural analysis of emulsions of different complexities, and the location of microorganisms in a wide range of food products. 

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