Isotope-enriched protein standards for computational amide I spectroscopy.

We present a systematic isotope labeling study of the protein G mutant NuG2b as a step toward the production of reliable, structurally stable, experimental standards for amide I infrared spectroscopic simulations. By introducing isotope enriched amino acids into a minimal growth medium during bacterial expression, we induce uniform labeling of the amide bonds following specific amino acids, avoiding the need for chemical peptide synthesis. We use experimental data to test several common amide I frequency maps and explore the influence of various factors on map performance. Comparison of the predicted absorption frequencies for the four maps tested with empirical assignments to our experimental spectra yields a root-mean-square error of 6-12 cm(-1), with outliers of at least 12 cm(-1) in all models. This means that the predictions may be useful for predicting general trends such as changes in hydrogen bonding configuration; however, for finer structural constraints or absolute frequency assignments, the models are unreliable. The results indicate the need for careful testing of existing literature maps and shed light on possible next steps for the development of quantitative spectral maps.