A two-dimensional view of the folding energy landscape of cytochrome c

Abstract

Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an approximately 30-micros time scale.

Fig. 1.

Normalized cross-correlation curves for Cyt c–TMR over a range of pH values. A coarse rainbow coloring scheme has been used. Acid-denatured proteins are colored red (pH < 2.9). From pH 2.9 to 3.2, the correlation curves are colored orange. From pH 3.2 to 3.8, the curves are colored green. Above pH 4.0, the correlation curves are colored blue. A five-point boxcar smooth has been applied to these data to aid visualization.

Fig. 2.

Equilibrium denaturation monitored by FCS. The diffusional correlation time vs. the sample pH is plotted on the left-hand axis. The right-hand axis converts this correlation time to a hydrodynamic radius. A sigmoidal fit to the data is overlaid.

Fig. 3.

Representative fluorescence lifetime decays and maximum entropy fits and residuals. (Upper) The fluorescence lifetime decay, P(k) distribution, fit, and residual to TCSPC data for the acid-denatured protein (pH = 2.19). Because two channels were used in the fluorescence cross-correlation measurements, two lifetime decays were collected for each protein sample. (Lower) Same as above, but pH = 5.76. At pH 5.76, the protein is folded, and the TMR is quenched by means of Förster transfer to the heme.

Fig. 4.

Evolution of folding reaction coordinates and potential energy surfaces as a function of pH. (Left) The P(k) distribution determined by maximum entropy as a function of pH. (Center) Conversion of the P(k) distribution to a P(r) distribution. Overlaid (vertical bars) is the average hydrodynamic radius determined by FCS. (Right) The evolution of the potential energy surface for the dye–heme separation distance. These surfaces were made assuming that the middle P(r) distribution was governed by a potential of mean force.

Fig. 5.

Correspondence of TCSPC and FCS data. (Top) Plot of the fraction of molecules in the intermediate state as determined by TCSPC. (Middle) Fraction of molecules that are flickering while crossing the probe volume. (Bottom) Time scale of this flicker. We attribute the fluorescence flicker to conformational fluctuations between the unfolded protein and a compact intermediate state (see Discussion for details).