DSC OF IMMOBILIZED PROTEINS

我们建议使用差示扫描量热法来研究去折叠 固定在固体载体上的蛋白质。 目标是扩展 DSC允许对展开的蛋白质进行热力学研究的能力 不可逆地溶解。 许多目前工人感兴趣的蛋白质 使用DSC不可逆地展开，限制或阻止定量分析 的数据。 不可逆的解折叠通常是由于聚合物的聚集。 未折叠的蛋白质分子。 众所周知，通过将蛋白质附着到 固体支持物，可以防止聚集并且更可能重折叠 发生。 我们建议表征蛋白质展开固体支持 如交联琼脂糖，并确定 对固定化蛋白质进行有用的DSC研究。 初步研究 将使用一组已被很好表征的基准蛋白质 并且已知在溶液中可逆地解折叠，即核糖核酸酶A，α- 胰凝乳蛋白酶、溶菌酶和肌红蛋白。 这些分子的热解折叠 蛋白质将在相同的条件下在溶液中和 固体载体。 糜蛋白酶将为该方法提供方便的检测 由于在溶液中展开的可逆性可以通过实验 通过调节离子强度来控制。 最后，我们打算 研究使用这种技术来研究蛋白质的解折叠 没有观察到其可逆地展开。 利息将 集中在两种对本实验室的研究至关重要的蛋白质上： 肌球蛋白亚片段-1和重肌球蛋白。 肌球蛋白亚片段-1含有 肌动蛋白结合位点和肌球蛋白的ATP酶活性位点，因此 是肌球蛋白头部的能量转换部分， 了解其功能的重要性，DSC是主要的 这些技术可以明确地提供证据， 域及其相互作用。 因此，有必要制定 允许球状肌球蛋白头可逆解折叠的方法。

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.