Chapter 11: In-cell NMR: Spectral Optimization and Population Adjustment by Rational Protein Engineering

The prospect of obtaining atomic-resolution data of proteins ‘at work’ in live cells, has spurred on many scientists to take their chances with NMR. In doing so, the most common disappointment is that the acquired in-cell NMR spectra turn out to be useless: the signal is poor, or even completely missing, because of excessive line broadening. However, this is no reason for despair as there are simple ways to tailor the protein to better serve your needs. In this chapter, we outline first the molecular factors that control line broadening in the cellular system, and show how these can be tuned at will by protein engineering to enhance the spectral properties. Second, we provide a specific example where rational mutation is used to make the folding equilibrium amenable to direct quantification of protein-stability changes in mammalian and bacterial cells.