Formation of local structure and overall chain dimensions in the 124-residue, four-disulfide protein bovine pancreatic ribonuclease A (RNase A) under conditions favoring either the native or partially folded states have been studied by nonradiative excitation energy transfer measurements. Three RNase A derivatives, doubly labeled with 2-naphthylalanine amide (fluorescent donor) at the C-terminus of each and 7-carboxymethylamino-4-methyl-coumarin (fluorescent acceptor) at the epsilon-amino group of lysine 1, 61, and 104, respectively [(1-124)RNase A, (61-124) RNase A, and(104-124) RNase A], were prepared. RNase A was modified by a two-step labeling strategy involving prior modification of the C-terminus with the donor probe by enzymatic methods, followed by modification of lysine epsilon-amino groups with the coumarin derivative. The derivatives were purified by liquid chromatography and characterized by tryptic mapping. The mono-labeled donor derivative (without acceptor) undergoes a reversible thermal folding transition (T-m = 48.3 degrees C; native RNase A, T-m = 54.4 degrees C), and all labeled derivatives retain enzymatic activity (activities against the substrate cCMP relative to native are 87 +/- 5%, 94 +/- 6.5%, 79 +/- 10%, and 207 +/- 15% for the donor-only and doubly-labeled derivatives with the acceptor at Lys 104, 61, and 1, respectively), supporting the suitability of these derivatives for protein folding studies. Time-resolved fluorescence measurements were used to determine the extent of nonradiative excitation energy transfer between donor and acceptor probes, which allowed recovery of parameters describing the distribution of interprobe distances and the diffusion coefficient of the ends of the segments defined by the pairs of sites labeled by the probes. Use of a donor with a relatively long intrinsic fluorescence decay rate allowed greater precision in the recovery of the interprobe diffusion coefficients compared with earlier studies using donors with shorter intrinsic decay rates, and this parameter provides an important measure of the extent of folding and degree of packing of the chain segments. Analyses for each derivative were carried out under solution conditions favoring native (pH 5.0, 22 degrees C, <0.7 M guanidinium hydrochloride) or denatured (>6 M guanidinium hydrochloride) chain conformations, both with and without intact disulfide bonds (in the absence or presence of dithiothreitol, respectively). For (1-124) RNase A in the native state, high interprobe diffusion rates and a broad interprobe distance distribution suggest flexibility of the N-terminal segment, in the native state in solution, that is not observed in the same segment of the unmodified molecule in the crystalline state analyzed by X-ray diffraction. At high (>6 M) guanidinium hydrochloride concentration in solution, addition of DTT resulted in an expected increase of the mean EED of the 64-residue segment in (61-124) RNase A, as a result of disulfide bond cleavage. The transfer of the molecule from high to low denaturant concentration with disulfides broken resulted in a contraction of overall chain dimensions [the EED decreased from 33.3 Angstrom to 22 Angstrom for the (104-124) segment and from > 50 Angstrom to 38 Angstrom for the (61-124) segment as a result of this transfer]. In conclusion, the measurements show that the unfolded state and the partially folded states are far from being describable as statistical coils. The current experiments show the effectiveness of long-range distance determination for investigating the structure and flexibility of partially folded conformations and the functional native state as well.
