Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles.
Sequence-specific assignments have been obtained for side chain methyl resonances of Val, Leu and Ile in the outer membrane protein X (OmpX) from Escherichia coli reconstituted in 60 kDa micelles in aqueous solution. Using previously established techniques, OmpX was uniformly 2H,13C,15N-labeled with selectively protonated Val-gamma(1,2), Leu-delta(1,2) and Ile-delta1 methyl groups. The thus labeled protein was studied with the novel experiments 3D (H)C(CC)-TOCSY-(CO)-[15N,1H]-TROSY and 3D H(C)(CC)-TOCSY-(CO)-[15N,1H]-TROSY. Compared to the corresponding conventional experimental schemes, the TROSY-type experiments yielded a sensitivity gain of about 2 at 500 MHz. The overall sensitivity of the experiments was further enhanced more than two-fold by the use of a cryoprobe. Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the delta1-methyls of Ile. The present approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to about 100 kDa, and adds to the potentialities of solution NMR for de novo structure determination as well as for functional studies, such as ligand screening with proteins in large structures.
Hilty
Christian
C
wuthrich@mol.biol.ethz.ch
Fernández
César
C
Wider
Gerhard
G
Wüthrich
Kurt
K
D000596:Amino Acids / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003080:Cold Temperature; D029968:Escherichia coli Proteins; D006867:Hydrolases; D007532:Isoleucine / Q000737:chemistry; D007554:Isotopes; D007930:Leucine / Q000737:chemistry; D008567:Membranes, Artificial; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010742:Phospholipid Ethers; D012680:Sensitivity and Specificity; D014633:Valine / Q000737:chemistry
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D007554:Isotopes; D008567:Membranes, Artificial; D008823:Micelles; D010742:Phospholipid Ethers; D007532:Isoleucine; C482905:OmpX protein, E coli; D006867:Hydrolases; D007930:Leucine; D014633:Valine