The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK). A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively. The latter was subsequently labeled with either 5- or 4-acetamidosalicylic acid (the acceptor). From the comparative analysis of AK crystal structures [Schulz, G.E., Müller, C.W., & Diederichs, K. (1990) J. Mol. Biol. 213, 627-630] (apo-AK,AK.AMP complex and AK.AP5A [P1,P5-di(adenosine-5') pentaphosphate] complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure. The domain closure reduces interdomain distances in a two-step manner. Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E. coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex. Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex. The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively. Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A. The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E. coli AK in solution.

The purpose of this investigation is to characterize the reduced state of RNase A (r-RNase A) in terms of (i) intramolecular distances, (ii) the sequence of formation of stable loops in the initial stages of folding, and (iii) the unfolding transitions induced by GdnHCl. This is accomplished by identifying specific subdomain structures and local and long-range interactions that direct the folding process of this protein and lead to the native fold and formation of the disulfide bonds. Eleven pairs of dispersed sites in the RNase A molecule were labeled with fluorescent donor and acceptor probes, and the distributions of intramolecular distances (IDDs) were determined by means of time-resolved dynamic nonradiative excitation energy transfer (TR-FRET) measurements. The mutants were designed to search for (a) a possible nonrandom fold of the backbone in the collapsed state and (b) possible loops stabilized by long-range interactions. It was found that, under folding conditions, (i) the labeled mutants of r-RNase A in refolding buffer (the R(N) state) exhibit features of specific (nonrandom) compact but very dispersed subdomain structures (indicated by short mean distances, broad IDDs, and a weak dependence of the mean distances on segment length), (ii) the backbone fold in the C-terminal beta-like portion of the molecule appears to adopt a native-like overall fold, (iii) the N-terminal alpha-like portion of the chain is separated from the C-terminal core by very large intramolecular distances, larger than those in the crystal structure, and (iv) perturbations by addition of GdnHCl reveal several conformational transitions in different sections of the chain. Addition of GdnHCl to the native disulfide-intact protein provided a reference state for the extent of expansion of intramolecular distances under denaturing conditions. In conclusion, r-RNase A under folding conditions (the R(N) state) is poised for the final folding step(s) with a native-like trace of the chain fold but a large separation between the two subdomains which is then decreased upon introduction of three of the four native disulfide cross-links.

A search for the topology of the chain folding of reduced bovine pancreatic trypsin inhibitor (BPTI), in unfolded and partially folded states, was done by means of time resolved dynamic nonradiative excitation energy transfer (ET) measurements. Four double labeled BPTI derivatives were used in which the donor was attached to the N-terminal arginine residue and the acceptor was specifically attached to one of the lysine residues. The four derivatives form a series of labeled backbone segments of increasing length spanning the full lengths of the BPTI chain: 15, 26, 41, and 46 residues. The intramolecular segmental end-to-end distance (EED) distributions were determined for all the derivatives by global analysis of the decay curves of both the donor and the acceptor in the reduced state, in low (0.5 M) guanidinium chloride (GuHCl) concentrations at pH 3.6 and 2.1 (R and A states, respectively). The results show that, in the partial folding conditions of low GuHCl concentration, reduced BPTI is in a compact state, but in this state the polypeptide chain is not in a condensed statistical coil conformation. Two distinct subpopulations were found for the four intramolecular EED distributions. One subpopulation was compact, with native-like EED distribution, while the second was unfolded. The pairs of sites, residues 1 and 26 and residues 1 and 46, showed close proximity in the dominant subpopulation. These contacts form two loops (probably collapsed): one consists of the first 26 residues, and the second comprises the full length of the chain from the N- to the C-terminal segments, which is in fact made up to two shorter loops (1-26 and 27-46). The N-terminal 15 residue segment was relaxed into statistical coil-like non-native conformation, in contrast to its extended conformation in the native state. The effect of temperature in the range of 2-60 degrees C was small; the folded subpopulations were stable over this range. These results show that in BPTI the compact conformations found under unfolding and partially folding conditions have native-like chain topology. Under the conditions of transition to partially folding conditions the compact conformation is stabilized, not only by the hydrophobic collapse and the local interaction but also by nonlocal interactions (NLIs). Few specific, very stable NLIs between the three segments which form the main structural elements of the native conformation direct the formation of native-like topology of the chain in the transition.(ABSTRACT TRUNCATED AT 250 WORDS)

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