NMR Structure Determination of a Membrane Protein with Two Transmembrane Helices in Micelles:  MerF of the Bacterial Mercury Detoxification System

• Published in: Biochemistry (Easton) Vol. 44; no. 13; pp. 5196 - 5206
• Main Authors: Howell, Stanley C, Mesleh, Michael F, Opella, Stanley J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05-04-2005
• Subjects: Amino Acid Sequence; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Hydrophobic and Hydrophilic Interactions; Inactivation, Metabolic; Mercury - metabolism; Micelles; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048095v

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.