Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in C-enriched media, with specific C-labeled amino acids added when protein expression is induced. Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.