Membrane proteins play key roles in the signaling networks which enable cells to sense their environment and respond appropriately. Membrane-bound receptors function to bind specific molecules in the external environment of the cell and then transmit a signal across the membrane which alters processes in the cytoplasm. These processes are critical to multicellular organisms: coordination of the functions of all the cells requires intercellular communication. The breakdown of such processes appears responsible for diseases such as cancer, which is thought to arise from mutations in receptors involved in controlling cell growth. The molecular details of transmembrane signaling are poorly understood, in part because of the difficulties inherent in biophysical studies of membrane proteins. The proposed research aims to apply an emerging biophysical tool especially suited to membrane proteins. solid state NMR, to the study of a relatively simple and well-studied receptor involved in bacterial chemotaxis, the aspartate receptor. The strategy is to use biochemical techniques to introduce the nonperturbing spectroscopic probes (13C and 15N) into the active sites of the receptor, in different signaling states and mutant proteins. NMR experiments will then test proposed models which include dimerization and conformational changes of the receptor, with the ultimate goal of determining the molecular details of the mechanisms of transmembrane signalling. Two general NMR approaches are proposed to map the local structure in the receptor in different signaling states and mutant proteins. Classic CPMAS experiments on uniformly and specifically 15N labeled samples will observe changes in chemical shifts and solvent exchange of particular resonances upon ligand binding and methylation (involved in adaptation of the receptor to an ongoing stimulus). This will serve both to develop more specific models for the mechanism of signalling by identifying regions of the protein involved in structural changes, and to test existing models, for instance by determining whether proposed dimerization restricts the solvent exchange of particular side chains. The other NMR approach employs the novel rotational resonance technique to measure selected distances between 13C nuclei as a test of specific models for structure and mechanism. The Asp binding site will be examined by measuring ligand conformation and ligand to protein distances to determine the effects of methylation and probe the mechanism of adaptation. Studies of the methylation region will measure distances within and between proposed a helices, as a test of the proposed structure and suggested conformational changes. Finally, measurement of contacts between the two transmembrane helices as a function of both methylation and ligand binding will investigate how the protein propagates structural changes across the membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM047601-02
Application #
3468827
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1992-05-01
Project End
1997-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Type
Schools of Arts and Sciences
DUNS #
153223151
City
Amherst
State
MA
Country
United States
Zip Code
01003
Sferdean, Fe C; Weis, Robert M; Thompson, Lynmarie K (2012) Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms. Biochemistry 51:6920-31
Fowler, Daniel J; Harris, Michael J; Thompson, Lynmarie K (2012) Heat management strategies for solid-state NMR of functional proteins. J Magn Reson 222:112-8
Fowler, Daniel J; Weis, Robert M; Thompson, Lynmarie K (2010) Kinase-active signaling complexes of bacterial chemoreceptors do not contain proposed receptor-receptor contacts observed in crystal structures. Biochemistry 49:1425-34
Fowler, Daniel J; Khalifah, Peter G; Thompson, Lynmarie K (2010) Design and characterization of a calixarene inclusion compound for calibration of long-range carbon-fluorine distance measurements by solid-state NMR. J Magn Reson 207:153-7