Broad-Band Ultraviolet CD Spectroscopy of Ultrafast Peptide Backbone Conformational Dynamics

Abstract

The far-UV spectral window widely used for the conformational analysis of biomolecules is not easily covered with broad-band lasers. This has made it difficult to use circular dichroism (CD) spectroscopy to directly follow fast structure changes. By combining transient CD spectroscopy in the deep-UV with thioamide substitution, we demonstrate a method to overcome this difficulty. We investigated a dipeptide whose two carbonyl oxygen atoms were replaced by sulfur, red-shifting the strong lowest-lying * transitions into the more accessible 250-370 nm spectral window. Coupling of the two thioamide units cannot be resolved by achiral 2D-UV spectroscopy, but it gives rise to a pronounced bisignate CD spectrum. The transient CD spectra reveal weakening of this coupling in the electronically excited state, where conformational constraints are released. Our results show that direct local probing of fast backbone conformational change via CD spectroscopy is possible in combination with site-selective thio substitution in peptides and proteins.

Ultrafast photoinduced energy and charge transfer

“…The time-resolved CD experiments reveal the weakening of this coupling upon 266 nm excitation, which releases conformational constraints. 226 These results show that direct local probing of fast backbone conformational change via CD is possible. Furthermore, site-selective thio-substitution promises a more detailed probing of conformational dynamics in peptides and proteins.…”

Recent Trends in Chiroptical Spectroscopy: Theory and Applications of Vibrational Circular Dichroism and Raman Optical Activity

“…[19] Even worse situa- tion is expected for microwave (rotational) CD, with expected g~10 À 5 -10 À 6 , which is one of the reasons why rotational CD has been explored only theoretically so far. [20] Vibrational Raman optical activity (ROA) is defined in an opposite way to CPL ("right minus left"), as DI ðROAÞ ¼ I R À I L (7) and in analogy to the g-factor, circular intensity difference is usually used, defined as…”

“…Outside the scope of this review, but worthy to mention, are detection techniques allowing to combine optical activity with time resolution of very fast processes. [6,7] We focus on the vibrational optical activity and associated experiments, which are perhaps less known than the electronic spectroscopy, in particular the omnipresent electronic circular dichroism, but which have been very dynamically evolving in the past years. Below, basic terms and quantities concerning absorption, emission and scattering of polarized light are introduced, and selected recent discoveries concerning the "classical" or newer chiroptical spectroscopies are discussed, perhaps indicating future directions of the field.…”

“…For example, some electronic transitions may lie in the “vibrational” region and be measurable with the infrared optics, natural and magnetic optical activity can be measured at the same time, etc. Outside the scope of this review, but worthy to mention, are detection techniques allowing to combine optical activity with time resolution of very fast processes …”

Two-dimensional UV spectroscopy: a new insight into the structure and dynamics of biomolecules

“…In summary, 2DUV has the potential to become a powerful method to detect the folding state and the folding/ unfolding dynamics in unlabelled peptides, complementary to the established NMR and 2DIR techniques as well as to the recently developed ultrafast UV circular dichroism, 114 which has also shown the ability to monitor conformational changes in a peptide in real time. 115…”