P-Nitrophenylalanine as a Novel Probe of Hydrogen Bonding in Peptide Hydrogels and Proteins

Abstract

Using vibrational probes to report on the local environment of specific protein residues has became very popular in recent years. Aromatic nitro groups are easily incorporated into peptides and proteins, and the vibrational modes of the nitro groups have been proposed as IR (or Raman) probes. Currently, quantitative data on how the nitro group's frequencies respond to the local environment is unclear. Previous IR work led to focus on a mode near 1600 cm-1. Now, we see a very strong Raman scattering peak at about 1350 cm-1: calculations indicate that this comes from a normal mode that contains symmetric nitro and ring motions. We used the compound 4-nitro-benzyl alcohol and a home built Raman spectrometer to determine the solvatochromism of this band in a wide range of solvents. Our results showed that strong hydrogen bond donor solvents led to a blue shift in the frequency of this Raman band, and a linear regression analysis using empirical solvent scales indicated that H-bond donors are the main factor that determines the nitro stretching frequency. Using this new information, we placed p-nitrophenylalanine as a Raman probe in a hydrogel-forming peptide derived from semenogelin I. Our results showed that the peptides stayed intact and the probe was clearly visible across different concentrations in both H2O and D2O, with different frequencies in three different positions where p-nitrophenylalanine replaced either phenylalanine or tyrosine residues along what appears to be a hydrophobic cluster that holds together the hydrogel's beta sheet superstructure. P-nitrophenylalanine was also incorporated into the E. Coli acyl carrier protein at key sites using the stop codon suppression technique. In all peptide and protein samples, this band is visible despite its spectral overlap with other vibrations.