Direct observation of tiers in the energy landscape of a chromoprotein: a single-molecule study

Abstract

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.

Fig. 1.

Single-molecule fluorescence-excitation spectroscopy. (A) X-ray structure of the peripheral LH2 complex from R. molischianum as determined by Koepke et al. (18) (Upper) and top view of the arrangement of the BChl a molecules in the pigment protein complex (Lower). The protein backbone has been omitted for clarity. The B800 (yellow) and B850 (red) chromophores are shown. (B) Fluorescence-excitation spectra of LH2 complexes from Rs. molischianum. The top traces show the comparison between a room temperature ensemble absorption spectrum (red) and the sum of 24 fluorescence-excitation spectra recorded from individual complexes (black). The lower three traces display spectra from individual LH2 complexes. Each spectrum has been averaged over all possible excitation polarizations. The vertical scale is valid for the lowest trace; all other traces were offset for clarity. All spectra were recorded at 1.4 K with an excitation intensity of 10 W/cm².

Fig. 2.

Large spectral changes of the B800 absorptions. (A) Time sequence of 256 consecutively recorded fluorescence-excitation spectra stacked on top of each other. The fluorescence intensity is indicated by the grayscale. (B) Average of the stack of spectra shown in A. (C) Intensity of the fluorescence as a function of time for the spectral features (a and a′), as displayed in A. (D) Autocorrelation (upper solid gray and black lines for a and a′, respectively) and cross-correlation (lower solid black line) of the absorptions a and a′.

Fig. 3.

Small spectral fluctuations of an absorption. (A) Stack of 256 fluorescence-excitation spectra, from a narrow spectral region of Fig. 2 A at an expanded view around the position of line a′. The fluorescence intensity is indicated by the grayscale. The average absorption line of all spectra is shown in Lower and shows a linewidth of 41.6 cm^–1 (full width at half maximum). (B) Stack of 172 fluorescence-excitation spectra that were obtained after fitting each individual scan to a Lorentzian profile and subsequently shifting the individual scans so that the peak positions of the fits coincided. The spectrum in Lower displays the average of all scans and features a width of 7.5 cm^–1 (full width at half maximum).

Fig. 4.

(A) Schematic illustration of three subsequent tiers of the potential energy hypersurface of a protein as a function of an arbitrary conformational coordinate. (B) Width of the spectral region that is covered by the spectral fluctuations of the chromophore within a certain time window, termed energetic span, versus the rate of these fluctuations in the three tiers that were found. For details see Discussion.

Fig. 5.

Part of the binding pocket for a B800 BChl a molecule in Rs. molischianum (blue, N; red, O; green, Mg). Dashed lines indicate likely hydrogen bonds and metal ligands at short distances (Å).