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. So far we have shown in vitro that the binding of CALNUC to Gai3 if it is true must be very weak. We are currently trying to characterize possible partners that might regulate this interaction. In order to be able to study larger complexes by NMR we developed and adapted new technology in the laboratory. We adopted new NMR experiments to study larger proteins as well as labeling procedures (deuteration) that was not previously available for our group. In addition we showed by careful experiments and controls that one can use scalar coupling in addition to chemical shift to map out molecular interactions. This enables one to probe allosteric process that was previously difficult to distinguish from chemical shift information alone. At the same time we also showed that thru the use of paramagnetic spin label one can probe weak and transient interaction. We illustrated a protocol where one can determine a bound conformation of an amino acid binding protein, glutamine binding protein (GlnBP), from a known free or apo conformation using paramagnetic label alone. We further showed in the case of glutamine free form of GlnBP, using extensive paramagnetic relaxation enhancement data, does not sample the close conformation in solution. This is in contrast to Maltose binding protein that seems to transiently sample its close conformation, with 5% population, in solution. Based on what we learned here we can extend this protocols to look at various weak interactions in proteins involved in cell signaling cascades. In parallel we also observed a unique change of scalar coupling due to the presence of paramagnetic label. We believe that one contribution is due to the polarization of the spin orbital due to the electron field at the nucleus. In addition a contribution thru relaxation mechanism due to the interference between the electron dipole and nuclear dipole can not be ruled out. This is currently an on going project to test whether one can take advantage of this for structural information. We have adapted the paramagnetic spin labeling technology that we learned to study protein complexes important for the regulation of actin polymerization. The first such complex structure that we solved was Capping protein and V-1 (Myotrophin). We showed that the V-1 binding site overlaps that of the actin barbed end on capping protein. This explains the regulation of actin polymerization by capping protein thru its binding site sequestration in the cell by V-1. We confirmed out finding by mutation studies on V-1 as well as capping protein that modulate their affinity. The second complex that we solved was between capping protein and a peptide corresponding to the CAH3a and CAH3b regions of CARMIL. We showed that the binding site of CARMIL on capping protein is extensive and there are no overlap between CARMIl and actin binding sites on capping protein. Interestingly CARMIL peptide doesn't adopt any secondary structure in the free as well as the bound forms. Furthermore we showed that in either of these two complexes the terminal "tentacles" of the capping protein are not involved. They have been implicated in the past as important for actin regulation. We further showed a more realistic model, based on our structure of the complex, where tight binding regions on CARMIL orient its polypeptide chain such that a "interference" loop between the CAH3a and CAH3b domains is positioned close to the basic patch where actin supposed to bind. This close proximity can explain the reduction of capping protein affinity to actin, but not a complete inhibition. In the future we will investigate the ternary complex of capping protein, V-1, and CARMIL. We developed a new method to characterize inter-domain motion. We applied this new approach to study the functional flexibility of a three domain modules of factor-H, which is a protein involved in immune signaling against host pathogens. We used residual dipolar coupling (rdc) measured by NMR. The rdc is an average quantity which reflects the ensemble population of structures in solution. We showed that there is a maximum of 20 degrees cone angle between these domains. We also used a shape empirical potential in the calculation to test our finding. The agreement to the rdc was worse when a shape potential is used to limit the amplitude of motion. This amplitude of motion can explain the conformation observed for this protein when it binds the target protein C3b. We are currently carrying out simulation to test stochastic diffusion under various interaction potential that can reproduce the observed amplitude of motion in factor-H. We concluded a study in which we probed, by NMR, the temperature dependent of amplitude of motion of protein backbone which is related to heat capacity. We used GlnBP in the free and substrate bound form which we have studied previously. We showed that certain sites in the protein backbone showed decrease flexibility as the temperature increased. These residues include those involved in substrate binding as well as those making up the hinge region of the protein that allow domain closure upon substrate binding. This behaviour could be correlated to our earlier finding, where this protein doesn't sample close conformation in the absence of substrate, unlike other members of the family. In addition we also showed that hydrophobic residues forming ring stacking and salt bridge surrounding them also decreased their flexibility as a function of increased temperature.

Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$1,131,954
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
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