Nanosecond circular dichroism spectral measurements: extension to the far-ultraviolet region

Abstract

The first measurements of time-resolved circular dichroism in the far-UV spectral region (far-UV-TRCD) with time resolution on the order of lO-'s are presented. The capability of making such measurements is demonstrated with ground-and excited-state CD spectra of (A)-Ru(bpy)3*+ between 190 and 290 nm as well as with CD spectra of a short peptide measured at different pHs. The properties of the far-UV-TRCD technique are discussed in detail, and comparisons between the static CD spectra obtained by this technique and those obtained by a conventional CD spectrometer are presented. Possible improvements to the current technique and application of the technique to resolve biophysical problems such as the kinetics of peptide helix+oil transition are also discussed.

Nanosecond time-resolved polarization spectroscopies: Tools for probing protein reaction mechanisms

“…Such time resolution was achieved in the 1980s when the TRCD technique advanced to measurements on the nanosecond and picosecond time scales [33, 34]. Although initially designed with single wavelength detection [33], nanosecond TRCD spectroscopy in this laboratory has evolved to include multichannel detection [35], other nanosecond polarization methods (TRMCD [16], ultrasensitive time-resolved LD (TRLD) [36], time-resolved optical rotatory dispersion (ORD) (TRORD) [37] and time-resolved magnetic optical rotatory dispersion (MORD) (TRMORD) [38, 39]), extension of the measurements from the visible into the far-UV region [40], faster time resolution (~10 ns) [41], and most recently, it has been coupled with a T-jump trigger [42]. Thus, since the mid-1980s, most submillisecond polarization experiments on biomolecules have been performed with the nanosecond time-resolved methods that were developed in this laboratory.…”

Flash Photolysis and Chemistry of Transients and Excited States

“…A probe of polarized light has been used in flash photolysis to investigate the circular dichroism (CD) 20 and magnetic circular dichroism (MCD) 21,22 of reaction intermediates and excited states. The technical difficulties of the experiment are the small difference between the respective extinction coefficients for the right, « R , and left, « L , circularly polarized light at a given wavelength and the small relative shift of the spectra obtained with each light polarization.…”

Probing Early Events in Ferrous Cytochrome c Folding with Time- Resolved Natural and Magnetic Circular Dichroism Spectroscopies.

“…Since it was initially coupled with stopped-flow and flash photolysis methods in 1974, the time resolution of optical CD measurements has improved from milliseconds to picosecond [7–13]. The evolution of nanosecond CD (TRCD) ellipsometry from single-wavelength to multi-wavelength measurements and the extension of these measurements into the far-UV spectral region and to time-resolved magnetic circular dichroism (TRMCD) have been particularly useful for the study of protein folding.…”

“…Since the first measurements of far-UV TRCD data in 1993 [13], the method has been applied to the study of a variety of biochemical systems such as the phytochrome photoreceptor [14] and myoglobin [15]. The first application in far-UV TRCD studies of protein folding was a 1998 investigation of rapid helix formation in the cytochrome c -CO photolysis system [16] developed previously by Roder and coworkers [17].…”