Received September 3, 2004

Revised Manuscript Received February 8, 2005

Abstract:

The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone ¹H-¹⁵N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the ¹H-¹⁵N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The ¹H-¹⁵N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 Å. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.

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