| A protein's structure, stability, and dynamics are frequently used to explain its function. To date, proteins are most often studied in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein characteristics such as stability and dynamics? The answer will help in assessing protein function in cells and illuminate ways of understanding and combating disease. To this end, a novel technique, 'in-cell' NMR, can be used to observe proteins inside living Escherichia coli. The object of this work was to develop this technique further to yield quantitative information about protein stability and dynamics in living cells.; Most of the work concentrates on the motions of the protein backbone. The backbone dynamics of apocytochrome b5 in living cells were quantified by making 15N relaxation measurements using in-cell NMR. The overall trend of backbone dynamics remains the same in cells. Most of the differences observed track the increased intracellular viscosity rather than a change in dynamics. Methods for measuring protein stability in cells were also designed and investigated, though the quantification of protein stability using in-cell NMR is on going. |
