5-Cyanotryptophan as an Infrared Probe of Local Hydration Status of Proteins

Abstract

The nitrile (C≡N) stretching vibration is sensitive to environment, making nitrile-derivatized amino acids an increasingly utilized tool to study various biological processes. Herein, we show that the bandwidth of the C≡N stretching vibration of 5-cyanotryptophan is particularly sensitive to water, rendering it an attractive infrared probe of local hydration status. We confirm the utility of this probe in biological applications by using it to examine how the hydration status of individual tryptophan sidechains of an antimicrobial peptide, indolicidin, changes upon peptide binding to model membranes. Furthermore, we show that p-cyanophenylalanine and 5-cyanotryptophan constitute a useful fluorescence energy transfer pair.

Figure 1

C≡N stretching bands of 5-cyanoindole in THF and a water/methanol mixture (95/5, v/v), as indicated.

Figure 2

C≡N stretching bands of Fmoc-L-Trp_CN in THF and a water/THF mixture (60/40, v/v), as indicated. The concentration of Fmoc-L-Trp_CN was estimated to be ~3.3 mM based on weight, and the optical pathlength was about 130 µm. For easy comparison, the spectrum measured in water/THF mixture has been scaled by a factor of 1.3.

Figure 3

C≡N stretching bands of Trp9/Trp_CN in THF, water, and DPC micelles, as indicated. The peptide concentration was ~2 mM in all solvents, except water where the concentration was not determined. For easy comparison, the spectra obtained in water and DPC micelles have been scaled, by a factor of 2.2 and 0.62, respectively.

Figure 4

Comparison of the C≡N stretching bands of Trp11/Trp_CN (red) and Trp9/Trp_CN (blue) in DPC micelles. The Trp9/Trp_CN data are identical to those used in Figure 3 and the Trp11/Trp_CN spectrum was scaled by a factor of 1.3.

Figure 5

Absorption spectrum of Ac-Trp_CN-NH₂ in water (black), fluorescence spectrum of an equimolar (~20 µM) aqueous solution of Phe_CN and Ac-Trp_CN-NH₂ (blue), and fluorescence spectrum of Phe_CN-Ala-Trp_CN-NH₂ (red) in water (~16 µM). For fluorescence measurements, the excitation wavelength was 232 nm.