Immunological Significance: NKG2D is an activating receptor found on several types of immune cells. Stressed cells express cell-surface proteins such as MIC-A that bind NKG2D, initiating an important cytotoxic immune response. A constitutively stabilized MIC-A, resulting in a more stable NKG2D--MIC-A interaction, would be an improved immunological staining reagent and a potential therapeutic agent that could downregulate natural killer cell activation in graft-vs.-host disease (GVHD). Hypothesis: Crystal structures have revealed that a disordered portion of the MIC-A surface folds upon NKG2D binding. The NKG2D--MIC-A interaction exhibits unusual thermodynamic characteristics, including a high heat capacity, that are hypothesized to be related to this structural transition. Method: Undergraduate student researchers will test this hypothesis by selecting a strategy to mutate MIC-A according to the analysis of a computational algorithm, with the ultimate aim to produce a constitutively ordered MIC-A. We will then study the impact of these mutations on NKG2D--MIC-A stability, entropy, enthalpy, heat capacity, and kinetics using a surface plasmon resonance technique previously demonstrated for both TCR-MHC and NKG2D-ligand interactions. A more stable MIC-A loop region is predicted to result in lower heat capacity for the NKG2DuMIC -A interaction, but should not markedly affect dissociation rate. Teaching Aims: Students will learn standard protein manipulation and production techniques in the course of discovering new relationships between protein-protein interface structure, design, and thermodynamics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15AI058972-01
Application #
6754607
Study Section
Special Emphasis Panel (ZRG1-F07 (20))
Program Officer
Prograis, Lawrence J
Project Start
2004-06-01
Project End
2008-05-31
Budget Start
2004-06-01
Budget End
2008-05-31
Support Year
1
Fiscal Year
2004
Total Cost
$217,500
Indirect Cost
Name
Seattle Pacific University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
071853477
City
Seattle
State
WA
Country
United States
Zip Code
98119
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Peterson, Megan J; Snyder, W Kalani; Westerman, Shelley et al. (2011) Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence. J Chem Educ 88:986-989
Culpepper, David J; Maddox, Michael K; Caldwell, Andrew B et al. (2011) Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions. Mol Immunol 48:516-23
Mayer, Chad L; Snyder, W Kalani; Swietlicka, Monika A et al. (2009) Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies. BMC Res Notes 2:135