DSC OF IMMOBILIZED PROTEINS

点击翻译按钮获取中文摘要

We propose to use differential scanning calorimetry to study the unfolding of proteins immobilized on solid supports. The goal is to extend the capabilities of DSC to allow thermodynamic studies of proteins which unfold irreversibly in solution. Many proteins of present interest to workers using DSC unfold irreversibly, limiting or preventing quantitative analysis of the data. Irreversible unfolding is often due to aggregation of the unfolded protein molecules. It is well known that by attaching proteins to solid supports, aggregation can be prevented and refolding is more likely to occur. We propose to characterize protein unfolding on solid supports such as crosslinked agarose and determine the conditions necessary for performing useful DSC studies of immobilized proteins. Initial studies will be with a set of benchmark proteins which have been well characterized and are known to unfold reversibly in solution, i.e. ribonuclease A, alpha- chymotrypsin, lysozyme, and myoglobin. The thermal unfolding of these proteins will be studied under identical conditions in solution and on solid supports. Chymotrypsin will provide a convenient test of the method since the reversibility of unfolding in solution can be experimentally controlled with adjustment of ionic strength. Finally, we intend to investigate the use of this technique to study the unfolding of proteins which have not been observed to unfold reversibly. Interest will be centered on two proteins of primary importance to research in this lab: myosin subfragment-1 and heavy meromyosin. Myosin subfragment-1 contains the actin binding site and the ATPase active site of myosin and therefore is the energy transduction portion of the myosin head are of great importance in understanding its function, and DSC is one of the primary techniques which can unequivocally provide evidence on the existence of domains and their interactions. It is therefore necessary to develop methods permitting reversible unfolding of the globular myosin heads.