The slow folding reaction of barstar: The core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has be en studied in the presence of 2 M urea at 10 degrees C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compac t form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and i ts mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fraction al changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, an d 16 %. The product of the fast phase can bind the hydrophobic dye ANS. The se observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state second ary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible pa tches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediat e: (i) fast (tau(r) approximate to 1 ns) local motion of its indole side-ch ain, and (ii) a slower (tau(r) approximate to 7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in th e fully folded protein where it is completely buried in the hydrophobic cor e, suggests that the core of the intermediate is still poorly packed. The g lobal tumbling time of the fully folded protein is faster at 5.6 ns, sugges ting that the volume of the intermediate is 25 % more than that of the full y folded protein. The rate of folding of this intermediate to the native st ate, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0 .07(+/-0.01) min(-1) This rate compares to a rate of folding of 0.03(+/-0.0 05) min(-1), determined by double-jump experiments which monitor directly f ormation of native protein; and to a rate of folding of 0.05 min-l, when de termined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5.6 ns as the protein folds. On the other hand, the amplitude of t he short correlation time decreases rapidly with a rate of 0.24(+/-0.06) mi n(-1). These results suggest that tight packing of residues in the hydropho bic core occurs relatively early during the observable slow folding reactio n, before substantial secondary and tertiary structure formation and before final compaction of the protein. (C) 2000 Academic Press.