Dynamics and folding of a membrane protein and transcription activator

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A lipid membrane surrounds all animal, plant, and bacterial cells. Protein channels and receptors embedded in the lipid precisely control the flow of nutrients, waste products, energy, and information into and out of cells, but are poorly understood. Our long-term goal is to develop methods to overcome the many difficulties in membrane protein structure, folding and dynamics analysis. Recently, we have made progress in studying the folding of a bacterial membrane protein, the glycerol facilitator in detergent environments. We propose to continue this work by applying a new method for synthesizing the protein using cellular extracts and studying folding of the protein in a lipid membrane environment. Cell-free synthesis opens up the possibility of labelling the protein with isotopes for analysis by Nuclear Magnetic Resonance (NMR) spectroscopy, a method analogous to Magnetic Resonance Imaging (MRI). With labeled protein, we will study the detailed atomic dynamics of the protein that are key to understanding its biological transport mechanism. We will also continue our work along similar lines on the fungal antibiotic peptide alamethicin that is a model membrane protein. All lentiviruses, including the Human Immunodeficiency Virus (HIV), produce a regulatory protein called the Transactivator of transcription (Tat) that is an essential component of the machinery that permits the virus to replicate itself. By studying the detailed dynamics of a fragment of the protein we discovered that Tat is intrinsically disordered under physiological conditions and undergoes a small disorder-to-order transition during the expression of its biological activity. We now propose to study the dynamics of the entire protein using NMR spectroscopy and to extend the range of times over which dynamics will be studied in order to better characterize the unbound protein. We will also continue our studies characterizing the binding of the protein to another important cellular protein, calmodulin, and to RNA. Since Tat is a key player in the regulation of viral replication and viral latency understanding the dynamics and folding of Tat may provide the foundation for improved therapies.