Fluorine NMR of proteins

Abstract The research described in this review indicates the very wide scope of fluorine NMR in the examination of proteins, with applications ranging from studies of isolated proteins and the complexes of proteins with small molecules, to proteins in systems as complicated as living organisms. Some conclusions and themes that emerge from consideration of this work are the following. 1. (a) There are many examples of the use of fluorine observations to probe protein structure and the dynamics of structural changes. These efforts have provided information about proteins that would be much more difficult to obtain by proton NMR observations, even at 750 MHz. The presence of two slowly interconverting forms of the D-galactose sensory receptor, the role of intermediates in the unfolding of the rat intestinal fatty acid binding protein, the sensitivity of the structure of Che Y to phosphorylation, or the global structural alterations produced by a single amino acid change or addition observed in many systems, are aspects of protein structure and dynamics that may well be manifest in PMR spectra only as an unexplained line broadening, if they are detectable at all. 2. (b) The fluorine-labeling approach can be useful with materials in excess of 300 kDa, and in systems that are oligomeric (actin fibers) or polymeric (collagen). 3. (c) At the present stage of development fluorine NMR experiments with fluorinated proteins cannot provide the highly detailed, global structural information that is obtainable by proton NMR studies. However, primarily because of the high sensitivity of fluorine shielding to molecular environment, fluorine observations have very high sensitivity to subtle structural changes that arise from mutations, interaction of proteins with ligands, or changes in solution variables such as pH or ionic strength. 4. (d) Due to the high sensitivity of fluorine shielding to molecular environment, it is more likely than would be the case with proton NMR observations that processes that interconvert protein structures or involve ligand binding will be in the slow exchange regime, such that separate resonances are observed for the interconverting species. The possibility of determining the amount of each species present and obtaining the rates of their interconversion by standard NMR experiments is much higher in the case of the fluorine-labeled system. In studies of the interaction of fluorine-substituted benzenesulfonamide inhibitors of carbonic anhydrase these types of information were obtainable rapidly. By comparison, proton NMR studies of the interaction of benzenesulfonamide with the same enzyme were tedious and time consuming because so much effort had to be invested in interpreting spectra with many overlapping resonances. 5. (e) Fluorine observations are particularly useful in multicomponent systems. Without specific isotope labeling detailed NMR studies based on the spectroscopy of proton, carbon-13, or nitrogen-15 nuclei would be challenging for systems such as proteins constituted into micelles or other membrane models, and in experiments with intact cells and whole organisms. The examination of the gene 8 protein in micelles and studies of phosphoglucomutase production and activity in vivo are examples of situations where the extreme specificity of fluorine-labeling protocols and the lack of background signals from endogenous nuclei are used to advantage. 6. (f) Fluorine NMR experiments have provided much unique information about enzyme mechanisms, including the identification of intermediates, the elucidation of the similarities of protein subunits within an enzyme complex, and demonstrations of subunit interactions, that would have been difficult to obtain or would be unconvincing if based on proton NMR observations. Thus, fluorine NMR studies of phosphoglucomutase and thymidylate synthetase clearly show the presence of intermediates during catalysis that could only be inferred by other experiments, and the nature of subunit interactions in alcohol dehydrogenase or the cyclic AMP-binding protein from E. coli is straightforwardly illuminated by the fluorine-labeling technology. 7. (g) Chemical synthetic and biosynthetic methods can be found to produce nearly any desired fluorine labeled material for examination by NMR. Many pharmaceuticals and other compounds of commercial importance contain fluorine, and examination of their interactions with isolated proteins from living systems or intact organisms by fluorine NMR seems particularly appropriate. As in all areas of NMR spectroscopy, the application of fluorine NMR to the study of proteins is ultimately limited by the inherent sensitivity of the instruments used. Progress on this front as a result of increased magnetic field strengths, the use of high-temperature superconducting materials in probe components, and other technologies yet to be created is to be expected, and will continue to broaden the range of productive utilization of the fluorine labeling idea in studies of biological systems.