Lee, Jennifer Chen (2002) Mapping heme protein folding landscapes. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08202004-114236
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The folding dynamics of two four-helix bundles, Fe'-cytochrome b[...] (E. coli) and Fe'-cytochrome c' (R.palustris), have been investigated: in each case, folding was triggered by electron transfer (ET) to the unfolded Fe'-protein and probed by transient absorption spectroscopy. Although these two proteins are topologically homologous, they display strikingly different folding kinetics: for FE'-cyt [...], the extrapolated time constant in the absence of denaturant [...] is near the predicted value for intrachain diffusion; in contrast, Fe'-cyt c' folding is heterogeneous over a ms-s time range.
Nonnative methionine ligands in Fe'-cyt c' contribute to the complex folding kinetics. Removal of either Met-15 or Met-25 leads to simpler kinetics than found in the pseudo-wild-type (Q1A) protein. The amplitude of the intermediate folding phase ([...]) is diminished, suggesting that formation of near-native contacts may facilitate folding.
Cyt c' has a wild-type Trp-72 residue that can be exploited in fluorescence energy transfer (FET) kinetics measurements. Also, we have crystallographically characterized [...] a Trp-mutant, Q1A/F32W/W72F, in order to probe distances between position-32 and the heme during folding. Analysis of FET kinetics from Trp-72 and Trp-32 shows that there is structural heterogeneity in the denatured protein. Even at high guanidine hydrochloride (GuHCl) concentrations, 50% of the polypeptides adopt compact conformations (25-35A) in cyt c'. A burst phase [...] is revealed when stopped-flow triggered refolding is probed by Trp fluorescence intensity: measurements on the Q1A protein show that 75% of the Trp-72 fluorescence (83% for Trp-32, Q1A/F32W/W72F) is quenched in the mixing deadtime, suggesting that most of the polypeptides have collapsed.
Electronically excited Zn-porphyrin in GuHCl-denatured Zn-substituted cytochrome c (Zn-cyt c) reduces [...] about ten times faster than when embedded in the folded protein. Measurements of ET kinetics during folding reveal a burst intermediate in which one-third of the ensemble has a protected Zn-porphyrin and slow ET kinetics; the remaining fraction exhibits fast ET characteristic of a solvent-exposed cofactor. Importantly, our FET (cyt c') and ET (Zn-cyt c) experiments show that collapsed nonnative structures are not substantially more stable than extended polypeptide conformations.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||28 May 2002|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||20 Aug 2004|
|Last Modified:||26 Dec 2012 02:57|
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