Time Resolved Transient Circular Dichroism Spectroscopy Using Synchrotron Natural Polarisation

Abstract

<a> </a><p><a></a><a>Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative</a> approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarisation of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130 Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomers concentration changes of an azopeptide after a photoisomerisation. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterisation of biological processes, such as protein folding, protein-ligand binding.<a></a></p>

Mechanisms of Biomolecular Self‐Assembly Investigated Through In Situ Observations of Structures and Dynamics

“…[45,46] In addition to quantifying the concentration of a given analyte, spectroscopic methods report on changes in the molecular conformation, [46] environment, [30] and bonding [47] of the analyte. Optical spectroscopy methods may also be enhanced by coupling them with other tools such as pumpprobe, [23] stopped-flow, [47] or confocal microscopy. [30]…”

“…[68,69] Recently, Auvray et al have made significant progress in utilizing the natural polarization present at synchrotrons for CD measurements. [23] The authors measured photoisomerization events in an azobenzene-modified peptide during alternating pulses of light exposure to trigger cis-to-trans transitions and decreased the temporal resolution of broadband (120 nm to 600 nm) CD measurements by several orders of magnitude (10 mHz to 130 HZ). This allowed CD spectra to be collected with 520 μs temporal resolution (Figure 5a-d).…”

“…Fluorophores may either be covalently bound or adsorbed to the biomolecule undergoing selfassembly. Conventional fluorescence measures the emission [23] ).…”

“…A 2D representation of the data in (b) shows the change in peaks at 198 nm and 212 nm indicating changes to the secondary structure of the peptide as a result of cis to trans isomerization, which occurs after the pulse at 2.5 s. (c) Schematic of conformational changes to the FK-11-X peptide because of azobenzene photoisomerization. (d) Changes in CD at 190 nm (x) and 204 nm (+) that demonstrate the reversibility of conformational changes (adapted with permission from Auvray et al[23] ).…”

Multiscale conformational dynamics probed by time‐resolved circular dichroism from seconds to picoseconds

“…Indeed, comparing the full CD spectra before and after photoexcitation and using standard techniques such as SVD will be very informative in future experiments. In this respect, use of Synchrotron Radiation is very promising [29].…”