We have also initiated a structural study of a calcium binding protein, CALNUC. This protein in the calcium loaded state binds Galpha (Ga) 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 hypothesize that there are several different modes of binding to the Galpha. These different modes govern a subset of different functions that the Galpha would undertake to respond to a certain stimulus. We have constructed the CALNUC plasmid which encompasses the two EF hands. We now have the structure of the calcium binding domain of CALNUC. it posseses a typical calcium binding loop. We are characterizing its calcium binding and try to correlate binding affinity to its binding loop structure. The backbone dynamics of this protein has been measured and we're in the process of correlating that to function of this protein, specifically its Ga interaction. We hope to be able to deduce from the structure of CALNUC its specific function. So far from our calcium binding experiments we believe that its function is to buffer calcium, due to the lower calcium afinity relative to other calcium binding proteins that are associated with signaling. Interestingly CALNUC does interact with Ga. We are trying to express and purify Gai to study its specific interaction with CALNUC. We succesfully solved the structure of CALNUC. We showed that the protein does bind 2 calciums. We also determined that both bonding sites have similar binding affinity. The protein undergoes an unfolding event when the calciums are removed. This is unique for calcium binding protein family and we hypothesize that this is correlated to the function of the protein as calcium signaling as well as buffering protein. We recently determined the affinity of CALNUC towards the C-terminal helix of Gai3. We employed polarization anisotropy. The dissociation constant is quite weak which is in agreement with what has been observed in cell competition assays. We are now trying to determine the affinity towards the full length Gai3, with the goal of studying structural determinants in the complex of these two proteins that define their role in signal regulation. Another protein that we're insterested in is AGS3. This seems to down regulate Ga response to cell signaling. So far we have been able to express the protein to acceptable level, however we have not been able to purify this protein to within acceptable level for NMR study.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL001048-08
Application #
7154353
Study Section
(LBC)
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2005
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
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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