RESUMO
Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.
Assuntos
Proteínas Fúngicas/química , Lipossomos/química , Proteínas de Membrana/química , Proteínas Mitocondriais/química , Neurospora crassa/química , Desdobramento de Proteína , Medição da Troca de Deutério , Ressonância Magnética Nuclear BiomolecularRESUMO
Intrinsically disordered proteins carry out many important functions in the cell. However, the lack of an ordered structure causes dramatic signal overlap and complicates the NMR-based characterization of their structure and dynamics. Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues), three prototypes of intrinsically disordered proteins, which bind to microtubules and play a key role in Alzheimer disease, can be obtained within 5 days by a combination of seven-dimensional NMR spectra with optimized methods for automatic assignment. Chemical shift differences between the three isoforms provide evidence for the global folding of Tau in solution.
