Membrane proteins take part in many important physiological functions, and constitute key drug targets. Structural studies of membrane proteins by X-ray crystallography or by NMR spectroscopy are much more difficult than for soluble proteins. Since real membrane systems are far too large for investigation by solution NMR experiments, membrane proteins are often reconstituted in detergent micelles. From these micellar systems, spectra can be obtained using TROSY (Arora and Tamm 2001; Fernández et al. 2001b; Fernández and Wüthrich 2003). Membrane proteins in detergent/lipid micelles yield fewer NMR resonances and thus less signal overlap than a globular protein of the corresponding molecular mass. Even though the detergent molecules may represent a large fraction of the large overall mass of the mixed micelles, proper isotope labeling, i.e., 13C,15N-labeling of the protein and/ or use of deuterated detergents, ensures that protein NMR signals can be detected with little or no interference from the signals of the detergent molecules.
Resonance Assignments and Collection of Structural Constraints for Membrane Proteins
All presently available NMR structures of larger integral membrane proteins have been determined using TROSY-based NMR techniques on uniformly 2H,13C,15N-labeled proteins in detergent micelles (Arora and Tamm 2001; Arora et al. 2001; Fernández et al. 2001a,b, 2004; Hwang et al. 2002). For these large structures, the advantages of TROSY are particularly remarkable when performing triple-resonance experiments for backbone resonance assignment, where the application of TROSY results in sensitivity gains of more than 1 order of magnitude, as illustrated in Fig. 5.4 for OmpX in 60-kDa DHPC micelles (OmpX/DHPC). Without the application of TROSY, only amide groups located in mobile loops in the OmpX structure were detected, e.g., Glyl6 (Fig. 5.4, panel D), whereas no signals could be detected from structured regions of the protein.
On the one hand, uniform 2H-labeling is required to make best use of the TROSY effect; on the other hand, extensive deuteration limits the obtainable structural information, e.g., NOEs can only be obtained from 15N-1H groups, ttus, in the case of membrane proteins only low-precision structures can be obtained for ^-barrel proteins (Fernández et al. 2001a), whereas for a-helical membrane proteins often only the secondary structure can be determined. With selective protonation of specific positions in the molecule, e.g., methyl groups, in a perdeuterated background, some of the NOEs can be recovered, resulting in greatly improved precision of the structure determination (Gardner et al. 1997; Gardner and Kay 1998). Sequence-specific assignment of the protonated groups can be obtained by through-bond correlation experiments to the assigned 15N-1H resonances, ttis strategy was applied with deuterated [13C,15N]-OmpX in DHPC micelles with protonation of the Val, Leu, and Ile(8l) methyl groups (Hilty et al. 2002). Subsequent analysis of the 3D 15N-resolved [1H,1H]-NOESY and 13C-resolved [1H,1H]-NOESY spectra yielded a fivefold increase of the number of NOE distance constraints and a concomitantly greatly improved precision of the NMR structure of OmpX/DHPC (Fernández et al. 2004).
3D Structure Determination
With availability of TROSY, the first NMR structures of larger integral membrane proteins were determined in the last few years (Arora and Tamm 2001; Arora et al. 2001; Fernández et al. 2001a, b, 2004; Hwang et al. 2002). tte architecture of these three E. coli outer-membrane proteins consists of an eight-stranded antiparallel ^-barrel, where sequentially successive ^-strands are connected by loops on the extracellular and the periplasmic sides, tte fold of the outer-membrane protein OmpX (148 residues) was obtained in DHPC micelles of about 60-kDa molecular mass (Fig. 5.9) (Fernández et al. 2001a, b, 2004; Hilty et al. 2002, 2003), the polypeptide backbone fold of the outer-membrane protein OmpA (177 residues) was determined in dodecylphosphocholine (DPC) micelles of 50-kDa molecular mass (Arora et al. 2001), and the backbone fold of the outer-membrane enzyme PagP (164 residues) was determined both in DPC and in «-octyl-P-d-glucoside micelles of size 50-60 kDa (Hwang et al. 2002).
tte NMR spectral properties of a-helical membrane proteins are less favorable than those of the P-barrel proteins, and so far structure determination has been limited to smaller proteins (fewer than 100 amino acid residues) containing one transmembrane helix or short protein fragments. Furthermore, an appropriate folding protocol is often not available for a-helical membrane proteins. Although for the membrane-associated a-helical 29-residue polypeptide hormone glucagon in DPC micelles the secondary structure was determined by NMR in the early 1980s (Braun et al. 1983; Wider 2003), NMR structure determination of a-helical membrane
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