Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor

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Abstract
The structure of BPTI and reduced BPTI in concentrated guanidinium HCI (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Inter probe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its ε-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1–15, is in an extended conformation (with an average interprobe distance of 34 ± 2 Å) in the native state. Upon unfolding by reduction with DTT or β-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced averageinterprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted β sheet element (residues 18–35), increased upon unfolding. At −30°C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 ±20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 ± 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even underdenaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded folded proteins and for search for folding initiation sites (FISs) and early folding intermediates.