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Site-directed solid-state NMR distance measurements test mechanisms of transmembrane signaling in bacterial chemotaxis receptors

Yael Sylvia Balazs, University of Massachusetts - Amherst

Abstract

The molecular mechanism of transmembrane signaling is unknown. Investigations have been hampered by the limits of current biophysical methods. Recently developed solid-state nuclear magnetic resonance (NMR) techniques provide a new approach for selective distance measurements probing structure and function of membrane proteins including ligand interactions. ^ The environmental signal transduced by the serine receptor of bacterial chemotaxis is initiated by the attractant molecule serine. We initially demonstrated the feasibility of direct internuclear distance measurements between the 15N-amino labeled serine ligand and phenylalanine 13C-carbonyl labeled receptor. The two 4.0 ± 0.2 Å distances measured from the serine receptor to the ligand match the distances observed in the crystal structure of the ligand-binding domain fragment of the homologous aspartate receptor. Demonstration of the structural similarity between the aspartate receptor and serine receptor instigated further investigations into the mechanism of ligand specificity. ^ To probe transmembrane signaling we developed a new constant time version of the rotational resonance solid-state NMR technique with improved reliability and efficiency. Combined with a site-directed strategy, this is a valuable and general tool for probing structure in large membrane proteins. A CO( i) to Cβ(i + 3) distance measurement along the periplasmic edge of the second membrane-spanning helix, provides a structural constraint for an unmapped region of the serine receptor. The 5.4 Å internuclear distance measured in the presence and in the absence of serine shows that any signaling mechanism must conserve the helical pitch of the second transmembrane domain. ^ To test abundant and conflicting models of transmembrane signaling we measured an inter-helical distance across the dimer interface in the transmembrane region of the receptor. Computer modeling of this distance predicted sensitivity to proposed long-range ligand-induced conformational changes including piston, scissors, and rotational motions. Ile measured 5.0 Å distance provides a valuable structural constraint of tertiary structure. Both ligand-free and -bound signaling states of the receptor show the same inter-helical distance, suggesting that conformational changes are not propagated into the transmembrane domain. This approach provides a means for testing proposed mechanisms and mapping conformational changes involved in transmembrane signaling. ^

Subject Area

Biology, Molecular|Biology, Microbiology|Chemistry, Analytical|Chemistry, Biochemistry|Chemistry, Physical|Biophysics, General

Recommended Citation

Yael Sylvia Balazs, "Site-directed solid-state NMR distance measurements test mechanisms of transmembrane signaling in bacterial chemotaxis receptors" (January 1, 1999). Doctoral Dissertations Available from Proquest. Paper AAI9932290.
http://scholarworks.umass.edu/dissertations/AAI9932290

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