Abstract
Recently it has been suggested that the C≡N stretching vibration of a tryptophan analog, 5-cyanotryptophan, could be used as an infrared probe of the local environment, especially the hydration status, of tryptophan residues in proteins. However, the factors that influence the frequency of this vibrational mode are not understood. To determine these factors, herein we carried out linear and nonlinear infrared measurements on the C≡N stretching vibration of the sidechain of 5-cyanotryptophan, 3-methyl-5-cyanoindole, in a series of protic and aprotic solvents. We found that while the C≡N stretching frequencies obtained in these solvents do not correlate well with any individual Kamlet-Taft solvent parameter, i.e., π* (polarizability), β (hydrogen bond accepting ability), and α (hydrogen bond donating ability), they do however, collapse on a straight line when plotted against σ = π* + β - α. This linear relationship provides a firm indication that both specific interactions, i.e., hydrogen-bonding interactions with the C≡N (through α) and indole N-H (through β) groups, and non-specific interactions with the molecule (through π*) work together to determine the C≡N stretching frequency, thus laying a quantitative framework for applying 5-cyanotryptophan to investigate the microscopic environment of proteins in a site-specific manner. Furthermore, two-dimensional and pump-probe infrared measurements revealed that a significant portion (∼31%) of the ground state bleach signal has a decay time constant of ∼12.3 ps, due to an additional vibrational relaxation channel, making it possible to use 5-cyanotryptophan to probe dynamics occurring on a timescale on the order of tens of picoseconds.