Heteronuclear Multidimensional NMR Spectroscopy of Solubilized Membrane Proteins: Resonance Assignment of Native Bacteriorhodopsin

摘要

Resonance assignment is an essential first step in all NMR spectroscopy investigations, independent of whether structural or dynamic information is extracted from the spectra. In proteins, resonance assignmet is performed by using the strategy of sequence-specific assignment, whereby the neighborhood of amino acid spin systems is established from the spectra. Pairs, triplets or longer stretches of spin systems are subsequently matched to the amino acid sequence and spin systems are thus assigned to amino acids. Originally, the assignment was accomplished by using homonuclear two-dimensional spectra (NOESY, DQF-COSY, TOCSY) and the method was limited to small proteins.~([1]) Presently, a sequence-specific assignment based on triple resonance techniques~([2]) that require proteins uniformly labled with carbon-13 and nitrogen-15~([3]) is typically preferred. The size of the proteins for which an assignment and a subsequent extraction of structural information can be achieved has thus been substantially increased. More recently, the size limit for proteins that can be studied by using solution-state NMR spectroscopy has been extended further by two novel developments, namely the deuteration of proteins~([4]) to enhance the relaxation properties of the carbon nuclei and the TROSY technique~([5]) to enhance the relaxation properties of the amide protons and nitrogen nuclei.