Dynamics of the core tryptophan during the formation of a productive molten globule intermediate of barstar

Abstract

The evolution of the nanosecond dynamics of the core tryptophan, Trp53, of barstar has been monitored during the induction of collapse and structure formation in the denatured D form at pH 12, by addition of increasing concentrations of the stabilizing salt Na₂SO₄. Time-resolved fluorescence methods have been used to monitor the dynamics of Trp53 in the intermediates that are populated during the salt-induced transition of the D form to the molten globule B form. The D form approximates a random coil and displays two rotational correlation times. A long rotational correlation time of 2.54 ns originates from segmental mobility, and a short correlation time of 0.26 ns originates from independent motion of the tryptophan side chain. Upon addition of ~0.1 M Na₂SO₄, the long rotational correlation time increases to ~6.4 ns, as the chain collapses and the segmental motions merge to reflect the global tumbling motion of a pre-molten globule P form. The P form exists as an expanded form with ~30% greater volume than the native (N) state. The persistence of an ~50% contribution to anisotropy decay by the short rotational correlation time suggests that the core of the P form is highly molten and permits free rotation of the Trp side chain. With increasing salt concentrations, tight core packing is achieved before secondary and tertiary structure formation is complete, an observation which agrees well with earlier kinetic folding studies. Thus, the equilibrium model developed here for describing the formation of structure during folding faithfully captures snapshots of transient kinetic intermediates observed on the folding pathway of barstar. A comparison of the refolding kinetics at pH 7, when refolding is initiated from the D, P, and B forms, suggests that formation of a collapsed state with a rigid core and ~30% secondary and tertiary structure, which presumably defines a coarse native-like topology, constitutes the intrinsic barrier in the folding of barstar.