Site-Specific Spectroscopic Reporters of the Local Electric Field, Hydration, Structure, and Dynamics of Biomolecules

Abstract

Elucidating the underlying molecular mechanisms of protein folding and function is a very exciting and active research area, but poses significant challenges. This is due in part to the fact that existing experimental techniques are incapable of capturing snapshots along the 'reaction coordinate' in question with both sufficient spatial and temporal resolutions. In this regard, recent years have seen increased interests and efforts in development and employment of site-specific probes to enhance the structural sensitivity of spectroscopic techniques in conformational and dynamical studies of biological molecules. In particular, the spectroscopic and chemical properties of nitriles, thiocyanates, and azides render these groups attractive for the interrogation of complex biochemical constructs and processes. Here, we review their signatures in vibrational, fluorescence and NMR spectra and their utility in the context of elucidating chemical structure and dynamics of protein and DNA molecules.

Figure 1

FTIR spectra (in the C≡N stretching region) of β-Ala_CN and p-Phe_CN in H₂O and Fmoc-β-Ala_CN and Fmoc-p-Phe_CN in THF, as indicated (reproduced from Ref. with permission).

Figure 2

The C≡N stretching bands of a calmodulin (CaM) binding peptide (MLCK_3CN) in the presence and absence of CaM, showing the sensitivity of this vibration to its environment (reproduced from Ref. with permission).

Figure 3

FTIR spectrum of the peptide Phe_CN-Ala-Trp_CN in THF at 20 °C, showing that the intensity of the C≡N stretching band of Trp_CN is larger than that of Phe_CN.

Figure 4

FTIR spectrum of the peptide Phe_CN-Ala-Phe_N3 in water at 20 °C, showing the relative oscillator strengths of nitrile and azide. The peptide concentration was ~2 mM and the optical pathlength was ~70 μm.