title: Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein.
abstract: The earliest kinetic folding events for (betaalpha)(8) barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (betaalpha)(8) barrel protein. Within 150 micros, the alpha-subunit of Trp synthase (alphaTS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 micros, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.forename: Elena
authors.initials: E
authors.lastname: Kondrashkina
authors.forename: Can
authors.initials: C
authors.lastname: Kayatekin
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel