Microsecond Subdomain Folding in Dihydrofolate Reductase

The characterization of microsecond dynamics in the folding of multi-subdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain alpha/beta/alpha sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Forster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 mu s of initiation of folding from the urea unfolded state. During the fastest observable similar to 550 mu s phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed. (C) 2011 Elsevier Ltd. All rights reserved.