Bacteriorhodopsin folds through a poorly organized transition state.

The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations

Dependence of the apparent proteolysis rate constants (<i>k</i>_p) on concentration of substrate proteins.

<p>(A) Proteolysis of DHFR by 80 µg/ml thermolysin. (B) Proteolysis of RNase H by 200 µg/ml thermolysin. (C) Proteolysis of DHFR by 40 µg/ml subtilisin. Error bars show the standard errors from curve-fitting to a first-order rate equation.</p

Effect of cleavage products on apparent proteolysis rate constants of DHFR by thrmolysin.

<p>Product was generated by incubating varying concentrations of DHFR with thermolysin until no intact DHFR was detectable by SDS-PAGE. Intact DHFR was then added to the resulting reaction to the final concentration of 0.050 mg/ml.</p

Determination of <i>k</i>₀ and <i>K</i>_i by global fitting.

<p>(A) Fractions of intact protein remaining after proteolysis of 50 (▪), 100 (△), 200 (▾), 300 (○), and 500 (•) µg/ml DHFR by 80 µg/ml thermolysin are shown with the global fitting by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111416#pone.0111416.e003" target="_blank">Eq. 2</a>. (B) Residuals are shown for fitting proteolysis of 50 µg/ml (•) or 500 µg/ml (○) DHFR by a first-order rate equation. Residuals are shown for fitting the proteolysis time courses predicted by global fitting parameters (solid lines in A) for 50 µg/ml (―) or 500 µg/ml (---) DHFR to a first-order rate equation.</p

Effect of intact DHFR on the rate of proteolysis of a peptide substrate by thermolysin and subtilisin.

<p>The rate of proteolysis of ABZ-Ala-Gly-Leu-Ala-NBA by thermolysin or subtilisin was determined in the presence and absence of intact DHFR. The rate constants (<i>k</i>_p) were determined by fitting the change of the fluorescence intensity to a first-order rate equation. Values reported are the average of three replicates. Standard deviations are shown in parenthesis.</p><p>Effect of intact DHFR on the rate of proteolysis of a peptide substrate by thermolysin and subtilisin.</p