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.

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

“…In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter. The advantages of using the C≡N stretching vibration of Phe CN are that (i) it is located in a spectrally uncongested region (2,000−2,400 cm −1 ), where water has a relatively low absorbance; (ii) it has a reasonably large extinction coefficient; and (iii) it is, in most cases, a simple transition that is decoupled from other vibrational modes of the molecule (41).…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…-N 3 [27,[49][50][51], or -C≡N [28,52] groups, many of which have been summarized in recent reviews [53,54]. Of course, with such labels one gives up one of the beauties of IR spectroscopy, i.e., one has to chemically modify the protein, but many of these IR labels are small perturbations in comparison to for example fluorescence labels.…”

“…The first is the asymmetric N 3 stretch vibration of azidohomoalanine (Aha) with a reasonably high extinction coefficient of 300-400 M −1 cm −1 [27] (note that the corresponding number is wrong in Ref. [53]). It is a methionine analog that can be incorporated into a protein using a Met auxotrophic mutant strategy [55].…”

Solvatochromism and the solvation structure of benzophenone

“…1 While the electronic Stark effect has traditionally served as the method of choice for a biomolecular systems, the vibrational Stark effect has recently enjoyed some popularity. 2 As the name implies, the vibrational Stark effect exploits the perturbation of a vibrationally active mode in the probe molecule, which is typically interrogated using infrared absorption spectroscopy. The absorption spectrum is obtained for a system in the presa) jchackett@vcu.edu ence and in the absence of an applied field, and the difference spectrum utilized to derive | μ|/h, termed the vibrational Stark tuning rate.…”

“…[12][13][14][15][16] While the aforementioned nitrile probes are effective and well characterized, 17,18 they comprise only a small region of chemical parameter space. In fact, other nitrile and nitrile-free probes including alcohols, azides, fluorides, nitrosyl, and carbonyl compounds have been investigated both experimentally 2,4,19,20 and theoretically. [21][22][23][24][25] Furthermore, the aforementioned studies have all utilized the Stark effect, with a few notable exceptions 9,10 in which the nitrile solvatochromic shift was calibrated against a standard series of solvents.…”