A series of site-specific fluorescently labeled BPTI derivatives prepared by nonselective acylation and chromatographic separations

Abstract

Investigation of conformational transitions and, in particular, the folding/unfolding transitions of globular proteins by means of excitation energy transfer measurements depends on the availability of protein derivatives carrying donor and acceptor probes at well-defined pairs of sites. A series of bovine pancreatic trypsin inhibitor (BPTI) derivatives, each labeled at one of the .epsilon.-amino groups, was prepared. This was achieved by a nonselective acylation reaction using 7-dimethyl-amino-coumarin-4-acetyl-N-hydroxysuccinimide ester (DACA-NHSIE) as a reagent yielding a mixture of products. The mixture was resolved by affinity chromatography and reversed phase high performance liquid chromatography (HPLC). Four derivatives were obtained, each carrying the probe at one of the four amino groups. Identification of site of labeling and determination of the purity of the products was achieved by HPLC-tryptic peptide mapping. The labeled derivatives are active and can undergo a reversible denaturation/renaturation cycle. The spectral characteristics of the probe make it a suitable acceptor in energy-transfer measurements. The advantage of the approach described here, namely nonselective reaction combined with efficient fractionation procedures for the preparation of site specifically labeled derivatives, is that each of the amino groups can be labeled in a simple procedure, thus allowing for a maximal number of labeling sites which cannot be achieved when site-directed reagents (e.g. specific particular protection) are used. The present method yields derivatives which are useful in energy transfer measurements for determination of intramolecular distances between labeled sites. The derivatives should be useful in the analysis of the mechanism of protein folding and the intermediate structures involved.