ABSTRACT
Of all the biophysical methods currently available for obtaining high-resolution structural information of biological macromolecules, nuclear magnetic resonance (NMR) is the only one that can provide this information in solution under nearphysiological conditions. Recently, we and others have extended the scope of NMR applications by investigating conformation and dynamics of biological macromolecules not only under near physiological conditions but also directly inside living cells [1-8]. These investigations are made possible by the noninvasive character of the NMR technique. The aim of these in-cell NMR experiments is not to determine three-dimensional structures. For a full structure determination, which takes many
days of measurement time, an
in vitro
sample of a purified protein provides far better conditions. The strength of in-cell NMR experiments lies in their ability to monitor changes in the conformation, binding status, and dynamic properties relative to the
in vitro
state of the macromolecule. The most frequently used biophysical technique employed to study proteins inside
cells is fluorescence. Labeling of macromolecules with fluorophores can reveal their intracellular localization, their rotational as well as translational diffusion, and, in the form of FRET measurements, also binding events. The enormous importance of fluorescence techniques in cell biology is based on their sensitivity, which even enables the observation of single macromolecules under certain conditions. Compared with fluorescence, NMR spectroscopy is very insensitive, requiring minimal concentrations of the investigated macromolecule in the micro-to millimolar range. However, the unique advantage of NMR spectroscopy over all other techniques is its ability to detect structural changes, measure dynamic parameters, and investigate binding events, with an unparalleled structural resolution. The only other technique that offers similarly detailed information is x-ray crystallography; however, it requires three-dimensional crystals and cannot be applied to investigations of macromolecules in cells.
