Nonlocal Interactions Stabilize Long Range Loops in the Initial Folding Intermediates of Reduced Bovine Pancreatic Trypsin Inhibitor

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 °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. These interactions form loops of the chain segments whose ends are held in close proximity by the strong NLIs. The stabilization of closed loops of chain segments can be a very effective early step in the folding of BPTI. These observations are interpreted as an indication that, in unfolded BPTI, stabilization of long range loops by specific NLIs can facilitate the accelerated folding into a native topology, within the early steps of the folding pathway. Formation of loops is a very effective mean of reducing chain entropy at the expense of a minimal number of very stable interactions.