ANALYSIS OF THE STRUCTURE OF RIBONUCLEASE-A IN NATIVE AND PARTIALLY DENATURED STATES BY TIME-RESOLVED NONRADIATIVE DYNAMIC EXCITATION-ENERGY TRANSFER BETWEEN SITE-SPECIFIC EXTRINSIC PROBES

Formation of local structure and overall chain dimensions in the 124-r esidue, four-disulfide protein bovine pancreatic ribonuclease A (RNase A) under conditions favoring either the native or partially folded st ates have been studied by nonradiative excitation energy transfer meas urements. Three RNase A derivatives, doubly labeled with 2-naphthylala nine amide (fluorescent donor) at the C-terminus of each and 7-carboxy methylamino-4-methyl-coumarin (fluorescent acceptor) at the epsilon-am ino 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 modifi ed by a two-step labeling strategy involving prior modification of the C-terminus with the donor probe by enzymatic methods, followed by mod ification of lysine epsilon-amino groups with the coumarin derivative. The derivatives were purified by liquid chromatography and characteri zed by tryptic mapping. The mono-labeled donor derivative (without acc eptor) undergoes a reversible thermal folding transition (T-m = 48.3 d egrees C; native RNase A, T-m = 54.4 degrees C), and all labeled deriv atives retain enzymatic activity (activities against the substrate cCM P relative to native are 87 +/- 5%, 94 +/- 6.5%, 79 +/- 10%, and 207 /- 15% for the donor-only and doubly-labeled derivatives with the acce ptor at Lys 104, 61, and 1, respectively), supporting the suitability of these derivatives for protein folding studies. Time-resolved fluore scence measurements were used to determine the extent of nonradiative excitation energy transfer between donor and acceptor probes, which al lowed recovery of parameters describing the distribution of interprobe distances and the diffusion coefficient of the ends of the segments d efined by the pairs of sites labeled by the probes. Use of a donor wit h a relatively long intrinsic fluorescence decay rate allowed greater precision in the recovery of the interprobe diffusion coefficients com pared with earlier studies using donors with shorter intrinsic decay r ates, and this parameter provides an important measure of the extent o f folding and degree of packing of the chain segments. Analyses for ea ch derivative were carried out under solution conditions favoring nati ve (pH 5.0, 22 degrees C, <0.7 M guanidinium hydrochloride) or denatur ed (>6 M guanidinium hydrochloride) chain conformations, both with and without intact disulfide bonds (in the absence or presence of dithiot hreitol, respectively). For (1-124) RNase A in the native state, high interprobe diffusion rates and a broad interprobe distance distributio n suggest flexibility of the N-terminal segment, in the native state i n solution, that is not observed in the same segment of the unmodified molecule in the crystalline state analyzed by X-ray diffraction. At h igh (>6 M) guanidinium hydrochloride concentration in solution, additi on of DTT resulted in an expected increase of the mean EED of the 64-r esidue segment in (61-124) RNase A, as a result of disulfide bond clea vage. The transfer of the molecule from high to low denaturant concent ration with disulfides broken resulted in a contraction of overall cha in 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 meas urements show that the unfolded state and the partially folded states are far from being describable as statistical coils. The current exper iments show the effectiveness of long-range distance determination for investigating the structure and flexibility of partially folded confo rmations and the functional native state as well.