title: Microsecond subdomain folding in dihydrofolate reductase.
abstract: The characterization of microsecond dynamics in the folding of multisubdomain 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 α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster 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 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs 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.
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. arai@bio.c.u-tokyo.ac.jp
authors.forename: Munehito
authors.initials: M
authors.lastname: Arai
authors.forename: Masahiro
authors.initials: M
authors.lastname: Iwakura
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D053838:Scattering, Small Angle; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014961:X-Ray Diffraction
chemical_list: D029968:Escherichia coli Proteins; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase