A general goal of our research in structural biology is to be able to select structural problems based on their biological significance. In practice, however, many systems chosen, in particular mammalian or human proteins, are difficult or impossible to make in a form suitable for structural studies by NMR. In our experience this is the most serious limitation of NMR structure determination. To overcome this problem has become the largest effort in our laboratory, and the success of our laboratory in solving many structures of proteins in the recent past was primarily due to the fact that we made serious efforts to improve our ways of protein production. Thus, we have made the production of labeled an well-behaving protein the goal of this component. We propose to develop novel strategies for producing well-behaving isotope labeled proteins in amounts needed for NMR spectroscopy.
In Specific Aim 1 we will explore the use of protein tags to enhance the solubility and stability of the systems to be studied. We will start from the attachment of a highly soluble protein domain (B1 domain of protein G) and will modify the tag for different purposes.
In Specific Aim 2 we will develop methods for producing properly folded and isotope-labeled membrane proteins. This is a major frontier in NMR spectroscopy and structural biology in general. Recently, we have made a breakthrough as we are able to over-express a G-protein coupled receptors in Pichia pastoris and have worked out procedure to label the receptor at reasonable cost.
In Specific Aim 3 we will develop novel methods for producing disulfide- rich and/or glycosylated proteins using E. coli strains with an oxidative cytoplasm, or Dictyostelium discoideum.
Specific Aim 4 is to apply these methods to test proteins that are challenging an represent model systems for these new expression methods.
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