Vibrational probes are provide a unique and useful tool for studying local environments in proteins using Raman and IR spectroscopy. The probe of interest in this work is a nitro group covalently attached to either the amino acid phenylalanine (creating the unnatural amino acid 4-nitrophenylalanine) or the amino acid tyrosine (creating the unnatural amino acid 3-nitrotyrosine). The aromatic nitro group has a very strong Raman signal at 1350 cm<sup>-1</sup> that, while possibly overlapping with other common vibrational signals, is so much stronger that it is clearly visible even in peptides and proteins. Using this signal, one can study a variety of things about the nitro group's environment such as solvent exposure, beta sheet formation, or degree of folding about a protein or amino acid.  Solvatochromic experiments indicate that the nitro frequency depends completely on the presence of local H-bond donor groups.<br/>The pH dependent protonation state of the amino acid 3-nitrotyrosine was studied using Raman scattering by observing the shape and shift of the nitro peak Systematic spectral shifts were observed, as well as a very large gain in the intensity of the nitro group’s Raman peak when 3-nitrotyrosine is deprotonated at higher pH (possibly due to resonant Raman enhancement through absorption of the Raman excitation beam by the colored tyrosine anion). A 13-residue peptide, including a 4-nitrophenylalanine probe in place of one of the tyrosine residues of the original sequence, was synthesized and purified.  This sequence forms a pH-responsive hydrogel consisting of a mixture of β-sheet and random coil as shown by IR spectroscopy. We are currently investigating if the nitro group can be used as a probe to report on degree of water exposure at specific sites in the peptide in the hydrogel state, and whether the presence of the polarizing nitro group perturbs the overall structure around the otherwise hydrophobic residues that it is attached to.

Using aromatic nitro groups as vibrational probes: 4-nitro-phenylalanine and 3-nitrotyrosine

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.