Time-Resolved Circular Dichroism Study of Protein Conformational Changes

Abstract

Circular dichroism (CD) is known to be a very sensitive probe of protein conformation, in particular in the far UV where characteristics signatures of secondary structures exist. We have developed new techniques aiming to measure CD in a pump-probe experiment and applied them to the study of the dynamics of conformational changes in proteins. Two techniques were implemented, either a straightforward probe polarization modulation technique or a more sophisticated pump-induced ellipticity measurement. Thorough study of the ultrafast dynamics following photodissociation of carboxy-myoglobin will be presented. Experiments in the visible (Soret band) as well as in the far-UV (helix band) display a strong transient CD feature. Thanks to a classical coupled oscillator calculation, we show that this signal comes from a distortion of the proximal histidine following the heme doming which relaxes on a 100 psec timescale. We have also introduced this transient CD technique in a T-jump experiment in a poly(glutamic acid) sample. A 6°C T-jump is obtained by directly exciting the water at 1.5 µm with a nanosecond pulse generated by a Nd:YAG pumped OPO. CD is measured at 225 nm on a microsecond timescale with a 12 ns time resolution. At pH 4.8, we observe a decrease of the CD signal with a 0.8 µs time constant that we assign to the dynamics of the alpha helix denaturation. This technique is expected to complement other time-resolved techniques based on IR absorption or fluorescence transfer with the advantage of being more straightforwardly quantitative in terms of protein helicity.