Observation of sub-100 ps conformational changes in photolyzed carbonmonoxy-myoglobin probed by time-resolved circular dichroism
Résumé
Conformational changes in proteins, which are known to play a paramount role in biophysical processes, are attracting much attention. For example, the change in carboxy-myoglobin (MbCO) after dissociation of the CO has recently been observed with a 100 ps time-resolution in a time-resolved X-Ray experiment. Shorter time resolution is however out of reach of such experiments. In order to investigate these processes on an ultrashort timescale, we have set up a time-resolved circular dichroism (CD) experiment in MbCO. The principle of the experiment is the following: after excitation with a pump beam, the CO-heme link breaks and a deoxy-heme structure appears very rapidly (< 1 ps). As the heme CD in the Soret region is very sensitive to the geometrical arrangement of the surrounding aromatic residues, measuring the change in the CD spectrum with time allows one to gain insight into the first steps of these conformational changes. The experiment is carried out on a 230 µM, pH 8.0 MbCO sample excited with a 400 nm pulse. The CD is measured across the Soret band as a function of time with a sub-picosecond resolution. After the initial drop in the CD due to the instantaneous electronic change of the heme, we observe a variation of the signal on a sub-100 picosecond timescale. In order to analyze these results, we have developed a calculation after Applequist's normal mode CD theory. Calculation of the contribution of the main residues to the rotational strength allows us to assign the observed signal to the stress provoked on the proximal histidine by the heme doming. This stress vanishes on a 100 ps timescale as the F-helix relaxes to its steady-state position. Extension of this experiment toward the far ultraviolet will provide a promising technique to investigate elementary changes in the secondary structure of proteins.