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|Title:||Direct observation of fast protein folding: Distinct nanosecond and microsecond events in the folding of apomyoglobin|
|Author(s):||Ballew, Richard Martin|
|Doctoral Committee Chair(s):||Gruebele, Martin|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The rapid refolding dynamics of horse apomyoglobin are followed by a new temperature-jump fluorescence technique on a nanosecond to 0.5 millisecond time scale in vitro. Collapse to a compact state is complete in under 20 microseconds under strongly-nativizing conditions. The intrinsic tryptophan fluorescence (residue 14 in the A alpha helix) serves as a local probe of the A-helix and the disposition of the H alpha helix. Methionine (residue 131 in the H-helix) quenching of tryptophan fluorescence at the interface of the A- and H-helices in the compact or native-like structure is exploited to observe directly collapse from a cold denatured state to a molten globule or compact, native-like state. Refolding is characterized by distinct nanosecond and microsecond phenomena (250 nanoseconds and 3.5 microseconds).
The two kinetic phases are characterized by opposite effects on the intrinsic tryptophan fluorescence lifetime. The effects on the fluorescence lifetime are interpreted as secondary and tertiary structure formation: the nanosecond phase is assigned to local collapse and alpha-helix formation, and the microsecond phase is attributed to the interaction of the A- and H-helices in the formation of a collapsed, compact structure. Even at low viscosities, the dependence of the early folding rate on solvent viscosity is in agreement with diffusive behavior, indicating solvent exposed protein motion during the collapse.
|Rights Information:||Copyright 1996 Ballew, Richard Martin|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9712195|
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