Downhill versus barrier-limited folding of BBL 2: mechanistic insights from kinetics of folding monitored by independent tryptophan probes

Neuweiler, Hannes and Sharpe, Timothy D. and Johnson, Christopher M. and Teufel, Daniel P. and Ferguson, Neil and Fersht, Alan R.. (2009) Downhill versus barrier-limited folding of BBL 2: mechanistic insights from kinetics of folding monitored by independent tryptophan probes. Journal of Molecular Biology, 387 (4). pp. 975-985.

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Official URL: http://edoc.unibas.ch/47494/

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Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of an alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Biophysics Facility (Sharpe)
UniBasel Contributors:Sharpe, Timothy
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:29 Nov 2017 09:07
Deposited On:29 Nov 2017 09:07

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