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1 Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA
2 Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA
(RECEIVED November 6, 2005; FINAL REVISION January 5, 2005; ACCEPTED January 5, 2005)
Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein–detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS–KcsA mixed micelles form an oblate spheroid with ~180 SDS molecules per channel. K+ ions bind to the micelle-solubilized channel with a KD of 3 ± 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.
Keywords: backbone assignment; detergent; global fold; membrane protein; NMR; NOE; potassium channel; secondary chemical shifts
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