Nmr Studies Of The Regulation Of Cell Signaling

National Heart, Lung, And Blood Institute

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Abstract

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.[unreadable] [unreadable] 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.[unreadable] [unreadable] 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.[unreadable] [unreadable] We developed methods to improve our ability to characterize this type study by NMR. We looked into possible deviations in J coupling measurements as well as chemical shifts. Typically one only measures the average of the chemical shift tensor and the full tensor was not possible to be measured in solution. We recently developed a way to measure chemical shift tensors in solution. In addition to atomic position that we obtained from NMR measurements the full chemical shift tensor provides us with variation in the atomic density distribution that is very sensitive to structural environment. We showed that in the case of carbonyl chemical shift tensor, hydrogen bond strength dominates two of the three tensor components. While for amide nitrogen, there are a lot more factor that contribute to the observed tensor. We will expand on this new technology to further study protein structure at a much more detailed level than previously possible.[unreadable] [unreadable] This previous year we invented a new method to align protein in a magnetic field. We used Colagen type I that we polymerize in the field to create a well ordered matrix into which we can diffuse protein that we would like to study. This gel material is table at very wide range of temperature and pH. More importantly it is stable under detergent environment allowing us to study membrane proteins. In addition we went further in studying chemical shift tensor. We decided to concentrate on changes of the tensor rather than the absolute values. This way we can isolate a single structural contribution to the chemical shift tensor changes. We used Glutamine Binding protein as a model system. We used the changes between the glutamine bound form of the protein and the free form. This has allowed us to look at hydrogen bond, dihedral angle, and packing influences on chemical shift tensor.