title: Site-directed rotational resonance solid-state NMR distance measurements probe structure and mechanism in the transmembrane domain of the serine bacterial chemoreceptor.
abstract: The serine receptor of bacterial chemotaxis is an ideal system in which to investigate the molecular mechanism of transmembrane signaling. Solid-state nuclear magnetic resonance (NMR) techniques such as rotational resonance provide a means for measuring local structure and ligand-induced structural changes in intact membrane proteins bound to native membrane vesicles. A general site-directed biosynthetic (13)C labeling strategy is used to direct the distance measurements to a specific site; the distance is measured between a unique Cys residue and a non-unique, low-abundance residue (Tyr or Phe). A (13)C-(13)C internuclear distance measurement from (13)CO(i) to (13)C beta(i + 3) at the periplasmic edge of the second membrane-spanning helix (TM2) of 5.1 +/- 0.2 A is consistent with the predicted alpha-helical structure and thus demonstrates an accurate long-distance rotational resonance measurement in the 120 kDa membrane-bound receptor. These measurements require a correction for the rotational resonance exchange between the multiple labels of the non-unique amino acid and the natural-abundance (13)C, which is critical to distance measurements in complex systems. A second (13)C-(13)C distance measurement between the transmembrane helices provides a high-resolution measurement of tertiary structure in the transmembrane region. The measured 5.0-5.3 A distance in the presence and absence of ligand is consistent with structural models for the transmembrane region and a proposed signaling mechanism in which ligand binding induces a 1.6 A translation of TM2. This approach can be used for additional measurements of the structure of the transmembrane region and to determine whether the ligand-induced motion is indeed propagated through the transmembrane helices.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-9336, USA.
authors.forename: Binumol
authors.initials: B
authors.lastname: Isaac
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Gallagher
authors.forename: Yael S
authors.initials: YS
authors.lastname: Balazs
authors.forename: Lynmarie K
authors.initials: LK
authors.lastname: Thompson
mesh_terms: D002247:Carbon Isotopes; D003545:Cysteine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017459:Receptors, Amino Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012399:Rotation; D012697:Serine Endopeptidases; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D008565:Membrane Proteins; D017459:Receptors, Amino Acid; C038039:serine receptor; D014443:Tyrosine; D010649:Phenylalanine; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D003545:Cysteine; D005998:Glycine