Time-resolved circular dichroism: Application to the study of conformal changes in biomolecules

Circular dichroism (CD) is known to be a very sensitive probe of the conformation of molecules and biomolecules. It is therefore tempting to implement CD in a pump-probe experiment in order to measure ultrarapid conformational changes which occur in photochemical processes. We present two technical developments of such time-resolved CD experiments. The first one relies on the modulation of the probe polarization from left to right circular whereas the second one measures the pump-induced ellipticity of the probe with a Babinet-Soleil compensator. Some applications are described and extension of these techniques towards the study of elementary protein folding processes is discussed.


Introduction
Circular dichroism (CD), the difference in absorption for a left or a right circularly polarized light, is with optical rotation a unique optical characteristics of chiral molecules.Because chirality is primarily a geometrical property, CD is in turn very sensitive to the conformation of molecules.This feature makes CD an attractive probe for stereochemistry and especially for the study of biomolecules [1].Taking advantage of our knowledge in ultrafast optics and nonlinear optical properties of chiral molecules, we have developed new experimental schemes allowing such time-resolved CD (TRCD) measurements.

Time-resolved CD
In order to achieve ultrafast measurements, pumpprobe experiments have been utilized for a long time and TRCD experiments are based on the same principle.A first intense light pulse (the "pump") is sent onto the sample so as to provoke a change in the molecules which is the monitored by a second, weak, delayed pulse (the "probe").Our experimental set-up is based on a 1 kHz, 150 fs Titanium-Sapphire laser.The pump pulses are most often obtained after frequency-doubling or tripling the output of the laser (400 nm or 267 nm, 200 nJ).On the other hand, extensive use of nonlinear optics for the generation of new frequencies in frequency-mixing stages or in optical parametric amplifiers allows us to have a very versatile source for the probe [2].Depending on the experiments, we use probe pulses tunable in the visible (400-500 nm) or in the UV (230-350 nm).Pump and probe pulses are focussed on the sample.The delay between the pump and the probe is computer-controlled and can be varied up to 1.5 ns.The sample consists in a fused-silica, 1 mm thick cuvette which is maintained in constant motion in order to avoid cumulative heating effects.For all the experiments, the sample concentration is chosen so that the optical density at the pump wavelength is of the order of unity.It corresponds to concentration in the range 100-300 µM.
In order to measure the CD of the probe, we have implemented two different techniques : modulating the probe-polarization and measuring the probe ellipticity wirh a Babinet-Soleil compensator.

Modulation of the probe polarization
The most straightforward technique is to modulate the probe polarization from right circular to left circular.In that case, CD is translated into a modulation of the probe transmission which can be extracted from the signal with a lock-in detection.In our case, the probe polarization is alternately right and left circularly-polarized thanks to a longitudinal Pockels cell on which a ≤ 1.5 kV voltage is applied.Deconvoluting the signal transmitted through the sample directly yields the absorption and the CD.Measurement is then carried out for various pump-probe delays.This technique is very straightforward but suffers from many artifacts.Indeed, it is very difficult to obtain perfectly circular polarizations and a default in the symmetry of the left and right polarizations yields artifactual signal which are indiscernible from the CD to be measured.To overcome this problem, we have developed a procedure to get a very precise alignment of the Pockels cell in order to obtain perfectly symmetrical circular polarizations [3].This technique has been successfully applied to the study of the conformational changes in the heme pocket of myoglobin within 100 ps after photoexcitation [4].Among the issues most studied by biophysicists, protein folding is of paramount importance.The fundamental mechanisms at stake in the formation of globular protein are still strongly debated and much work is currently developed to decipher folding or unfolding processes in small peptides or proteins [10].CombiningTRCD in the far UV with phototriggering of folding such as T-jump or photoinduced charge transfer could be an alternative technique to probe the formation of secondary or tertiary structures.

Fig. 2 .
Fig. 2. Time-resolved CD for the chromophore of B. Japonicum in the protein (OBIP) and in solution (OxyBP).
Such is the case in most enzymatic reactions or in transmembrane signalling for example.