Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements.

摘要: A series of four bovine pancreatic trypsin inhibitor (BPTI) derivatives, site specifically labeled by (2-methoxy-1-naphthyl)methyl (MNA) at the N-terminal amino group and [7-(dimethylamino)-coumarin-4-yl]acetyl (DA-coum) one epsilon-amino groups, was prepared. The N alpha-MNA-Arg1-N epsilon-DA-coum-Lysn-BPTI [(1-n)BPTI] (n = 15, 26, 41, 46), were purified affinity chromatography high-performance liquid (HPLC). homogeneity each derivative its labeling characterized HPLC tryptic peptide mapping. Nonradiative energy transfer from MNA (donor) to DA-coum (acceptor) measured monitoring donor emission acceptor excitation spectra. Transfer efficiencies between 45% 85% observed. fluorescence decay in MNA-BPTI, a without an acceptor, is monoexponential, with lifetime 6.8 +/- 0.15 ns. kinetics for derivatives both showed small deviation monoexponential shorter average lifetimes. Analysis experimental curves yielded detailed intramolecular distance distribution functions pair sites. averages calculated are close values expected known structure BPTI crystalline state. thus obtained suitable investigation conformational transitions protein localized changes such as those involved folding or unfolding transitions.