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

Area of Science:

• Biophysics
• Spectroscopy
• Structural Biology

Background:

• Determining protein 3D structure is advancing, but monitoring protein structure within live cells is challenging.
• Fourier transform infrared (FTIR) spectroscopy offers a potential method for in-cell structural analysis.

Purpose of the Study:

• To evaluate FTIR spectroscopy's utility for probing protein structure in live bacterial cells.
• To demonstrate site-specific conformational analysis of proteins within a cellular environment.

Main Methods:

• Utilized selective isotope enrichment (¹³C and ¹²C) in a reverse-labeling strategy for protein expression.
• Employed Fourier transform infrared (FTIR) spectroscopy for in-cell measurements.
• Combined FTIR data with site-directed mutagenesis for residue-level conformational analysis.

Main Results:

• FTIR difference spectra of recombinantly expressed NuG2b protein in live cells closely matched spectra of isolated proteins.
• The cellular environment was found to not perturb the protein's overall structure.
• Local conformation of individual amino acids, including contributions to α-helix or β-sheet structures, could be monitored.

Conclusions:

• FTIR spectroscopy is a viable technique for monitoring protein structure and conformation within live bacterial cells.
• The method allows for detailed analysis of protein structure without perturbation by the cellular environment.
• Site-specific conformational changes can be accurately detected using this approach.