Department of Biochemistry and Cell Biology
Kevin R. MacKenzie
NMR spectroscopy can provide information about the structure and dynamics of membrane proteins that are dissolved in detergent micelles or reconstituted into lipid bilayers. Detergent-solubilized membrane proteins can tumble freely on a nanosecond timescale, allowing the application of solution NMR methods to the collection and assignment of NMR spectra. However, the detergent environment may alter the conformation of the protein, so sample conditions need to be correlated with other measures of stability or activity. A lipid bilayer provides the protein with a physical and chemical environment most similar to a natural membrane, but the large size and slow tumbling of lipid bilayer vesicles require the use of solid-state NMR methods. While the upper limit on the size of the macromolecular complexes to which solution NMR methods can presently be applied has been pushed well past 50 kD, solid state methods for structure determination of membrane proteins are more technically challenging and are still being developed. The following reviews provide perspectives on the state-of-the-art in these endeavors.
R Fu and TA Cross Annu Rev Biophys Biomol Struct (1999) 28, 235-68
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SJ Opella Nat Struct Biol (1997), 4(5) Suppl, 845-8
Standard triple- or quadruple-resonance solution NMR methods enable the assignment of resonances, the determination of backbone and sidechain geometries using J couplings, and the measurement of inter-atomic distances using the nuclear Overhauser effect (NOE). These methods have been applied to several membrane protein systems, including the TM domain of glycophorin A, which forms a detergent-resistant parallel dimer of helices. While chemical shift dispersion of both backbone and sidechain resonances is poor and linewidths are generally broad, resulting in peak overlap, complexes containing at least four transmembrane helices should be analyzable by solution NMR if favorable detergent conditions can be identified.
The process of determining the structure of a complex of transmembrane helices might involve the following steps:
Biophysical investigations of macromolecules usually rely on the availability of a highly purified sample. The strongly hydrophobic nature of transmembrane domains of helical proteins necessitates the use of detergents or organic solvents; reverse-phase HPLC in organic solvents is a particularly useful purification method. The final samples will preferably be studied in detergent micelles. The choice of detergent and the path by which the protein is transferred from organic solvents into detergent micelles can greatly affect the structure and aggregation state of the TM domains, and hence their suitability for NMR structure determination.
Labeling the protein with stable isotopes 15N, 13C and/or 2H can greatly improve the quality of the NMR data that can be obtained. This is usually accomplished by expression in bacteria grown in labeled media, since chemical synthesis of uniformly isotopically labeled peptides is extremely expensive. However, chemical synthesis can provide a convenient route to the production of peptides with labels incorporated at discrete sites.