Journal of Biomolecular NMR

, Volume 27, Issue 4, pp 377–382 | Cite as

Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles

  • Christian Hilty
  • Gerhard Wider
  • César Fernández
  • Kurt Wüthrich

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.

membrane proteins OmpX solution NMR stereospecific assignment 

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Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Christian Hilty
    • 1
  • Gerhard Wider
    • 1
  • César Fernández
    • 1
  • Kurt Wüthrich
    • 1
  1. 1.Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule ZürichZürichSwitzerland

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