The interaction between the GTP-binding protein, its receptor, and its effector at the plasma membrane is well characterized. In contrast the specific interaction and function of similar system in the Golgi membranes is still not clear. A G Alpha Interacting Protein (GAIP) was chosen as a model to study this interaction. GAIP interacts specifically with the Gai3 which has been localized to the Golgi membranes. A plasmid construct containing the core domain (150 residues) of GAIP was constructed. The core domain of GAIP contains homology domain found in a novel family of regulators of G protein signaling (RGS proteins). The three dimensional fold of human GAIP has been determined using NMR spectroscopy. The refinement of the structure of GAIP is in progress. Human GAIP at concentration higher than 0.1 mM exists as a dimer in solution. This results in an effective MW of roughly 34 kD. The initial fold was determined without further deuteration of the protein which is typically done for structure determination of protein at this size by solution NMR. A backbone dynamic study of human GAIP has also been carried out using NMR. This confirms our finding that GAIP exists as a dimer in solution at least at concentration higher than 0.1mM. The dynamic data also reveals the regions which have flexibility. Initial comparison of GAIP and the X- ray structure of RGS4 complexed to Gai1 reveals some conformational changes upon binding to the G protein. The dynamic data suggests possible flexibility that allows the conformational change in the structure. A parallel project to express the G ai3 subunit has been initiated. The goal is to be able to reconstruct human GAIP and its G protein complement in vitro and observe the biochemical properties. We have completed the solution structure of human GAIP and carried out careful comparison to the structure of RGS4complexed to Galpha-i. We concluded that the activation of catalysis by RGS protein is through stabilization of the complex structure not by direct interaction of RGS to the active site of Galpha. Furthermore we have shown that the loop between helix Vand VI which contacts the Galpha differ in structure only for the N-terminal portion. The C-terminal portion of this loop does not adopt different conformation upon binding the Galpha. We are finishing the dynamic study of this protein. We have also initiated structural study of a calcium binding protein, CALNUC. This protein in the calcium loaded state binds Galpha in the Golgi. It is believed that CALNUC is regulated through its interaction with Galpha to modulate calcium concentration in the Golgi apparatus. CALNUC does not seem to effect the GTP hydrolysis in Galpha. Therefore we hypotesize that there are several different mode of bindings to the Galpha. These different modes govern a subset of different functions that the Galpha would undertake to response to a certain stimulus. - G-Protein, GAIP, Golgi, GTP, NMR, RGS protein, G-alpha, Cell signaling, CALNUC, Calcium binding

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL001048-02
Application #
6290391
Study Section
Special Emphasis Panel (LBC)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Burton, Robert A; Tjandra, Nico (2007) Residue-specific 13C'CSA tensor principal components for ubiquitin: correlation between tensor components and hydrogen bonding. J Am Chem Soc 129:1321-6
Tjandra, Nico; Suzuki, Motoshi; Chang, Shou-Lin (2007) Refinement of protein structure against non-redundant carbonyl 13C NMR relaxation. J Biomol NMR 38:243-53
Chen, Kang; Tjandra, Nico (2007) Top-down approach in protein RDC data analysis: de novo estimation of the alignment tensor. J Biomol NMR 38:303-13
Burton, Robert A; Tsurupa, Galina; Hantgan, Roy R et al. (2007) NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment. Biochemistry 46:8550-60
de Alba, Eva; Tjandra, Nico (2006) Interference between cross-correlated relaxation and the measurement of scalar and dipolar couplings by Quantitative J. J Biomol NMR 35:1-16
de Alba, Eva; Tjandra, Nico (2006) On the accurate measurement of amide one-bond 15N-1H couplings in proteins: effects of cross-correlated relaxation, selective pulses and dynamic frequency shifts. J Magn Reson 183:160-5
Burton, Robert A; Tjandra, Nico (2006) Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media. J Biomol NMR 35:249-59
Chang, Shou-Lin; Tjandra, Nico (2005) Temperature dependence of protein backbone motion from carbonyl 13C and amide 15N NMR relaxation. J Magn Reson 174:43-53
de Alba, Eva; Tjandra, Nico (2004) Structural studies on the Ca2+-binding domain of human nucleobindin (calnuc). Biochemistry 43:10039-49
Kuszewski, John; Schwieters, Charles D; Garrett, Daniel S et al. (2004) Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments. J Am Chem Soc 126:6258-73

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