Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy

Our recently developed in-cell NMR procedure now enables one to observe pro tein conformations inside living cells. Optimization of the technique demon strates that distinguishing the signals produced by a single protein specie s depends critically on protein overexpression levels and the correlation t ime in the cytoplasm. Less relevant is the selective incorporation of N-15. Poorly expressed proteins, insoluble proteins, and proteins that cannot tu mble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discus s limitations of the technique as well as prospects for future applications .