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Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of 19F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy. [ABSTRACT FROM AUTHOR]

The Ca2+-mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by 19F NMR spectroscopy of the transition of the Ca2+-free and -bound forms in Xenopus laevis oocytes at different Ca2+ levels. We find that the Ca2+-bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca2+ level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that 19F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants. [ABSTRACT FROM AUTHOR]