Evaluation of p-(¹³C,¹⁵N-Cyano)phenylalanine as an Extended Time Scale 2D IR Probe of Proteins

Abstract

Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(CN-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.

Extended timescale 2D IR probes of proteins: p-cyanoselenophenylalanine

“…51 In addition, the water solvent itself strongly absorbs the mid-IR pulses, leading to off-diagonal spectral features associated with water heating. 41 For CNPhe, the 2D spectra at T w of 10 ps (Fig. 4A) already show line width distortion due to reduction in signal strength as a result of the fast lifetime of CNPhe.…”

“…40,41 In H 2 O, the CN lifetime for 13 C 15 NPhe (8.7–10.5 ps) is ~2.0–2.5 fold longer than CNPhe (4.0–4.6 ps), whereas those for 13 CNPhe(3.4–5.0 ps) and C 15 NPhe (0.90–2.2 ps) are roughly the same or even shorter. These are fairly small changes in lifetime and do not correlate with the small changes in mass, which indicates that the increase in mass from isotopic substitution does not substantially decouple the intramolecular vibrational relaxation pathways.…”

Supramolecular self assembly of nanodrill-like structures for intracellular delivery

“…The masses of all CSPNs were in agreement with the theoretical values, which further ensured the purity and synthetic efficacy of SPPS (Figure.S3–S7). Further, the intensity of aromatic C-H stretching was found to be in ascending order from RT to 4F-RT, which can be attributed to the increasing number of Phe,[46] as evident from the FT-IR spectral data (Figure.S8).…”

Assessing the Effect of Hofmeister Anions on the Hydrogen-Bonding Strength of Water via Nitrile Stretching Frequency Shift

“…In the current study, we develop a simple yet powerful method to extract thermodynamic information about the H-bonding interactions of interest, which is based on the notion that the stretching vibrational frequency (ν CN ) of a cyano (CN) group depends on its H-bonding status. , The sensitivity of the ν CN of various nitriles to its local environment is well documented and studied. − For example, Getahun et al have shown that the ν CN of an unnatural amino acid, p -cyanophenylalanine (Phe CN ), is shifted from 2237.2 cm –1 in water to 2228.5 cm –1 in tetrahydrofuran (THF), and other studies, especially those involving peptides and proteins, ,,,− have further demonstrated that ν CN depends on the local microenvironment of Phe CN . Although previous studies clearly established that H-bond formation can change the stretching frequency of a CN group, there is no simple correlation that can be used in practice to interpret an observed ν CN in terms of the strength of the underlying H-bonding interactions. − Therefore, we aim to develop a method that can yield such information.…”