title: Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles.
abstract: In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D 15N- and 13C-resolved [1H,1H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional 13C labeling of OmpX and [13C,1H]-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument.
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006867:Hydrolases; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry*; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010742:Phospholipid Ethers / Q000737:chemistry*; D011487:Protein Conformation; D013237:Stereoisomerism
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D008823:Micelles; D010742:Phospholipid Ethers; C482905:OmpX protein, E coli; C051224:methyl radical; D006867:Hydrolases; D008697:Methane