MEASURING COLLAPSE KINETICS OF LAMBDA REPRESSOR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。蛋白质折叠通常会被自由能垒减慢，但蛋白质可以被设计成非常稳定，并在没有明显障碍的情况下快速折叠。这种超稳定蛋白质可以作为引入新功能的“支架”，制造“设计蛋白质”。我们正在研究这样一种不同寻常的蛋白质的结构形成，这就是工程的波长抑制子。虽然蛋白质折叠非常迅速，但通过在粘性溶液中冷却它，我们可以通过在Argonne的BioCAT光束线上进行时间分辨X射线散射来跟踪致密结构的形成，该光束线是进行蛋白质水相散射研究的最明亮的光源。我们已经对这种蛋白质和我们新的STOP-LOW装置进行了初步研究，以优化信号，并在2005年继续我们的三种蛋白质变体的研究，其中一种是野生型，一种包含D14A突变，第三种是A37G/A49G突变。这些被选择来覆盖一系列的稳定性和聚集倾向。对小蛋白质折叠的默认假设是，X射线散射、荧光和红外光谱将产生相同的折叠时间(即所谓的双态折叠)。我们认为，根据我们的设计，D14A突变体将违反这一假设，而其他两个突变体将符合这一假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein folding is usually slowed down by a free energy barrier, yet proteins can be designed to be very stable and to fold rapidly without a significant barrier. Such hyperstable proteins could serve as "scaffolds" for introducing new function and make "designer proteins." We are studying the formation of structure in one such unusual protein, engineered lambda repressor. Although the protein folds very rapidly, by cooling it down in viscous solutions, we can follow the formation of compact structure by time-resolved X-ray scattering at the BioCAT beamline at Argonne, the brightest source for aqueous protein scattering studies. We have conducted initial studies of the protein and our new stopped-low apparatus to optimize signals, and are continuing our studies in 2005 with three protein variants, the wildtype, one containing a D14A mutation, the third a A37G/A49G mutation. These were chosen to cover a range of stabilities and aggregation propensities. The default hypothesis for the folding of small proteins is that X-ray scattering, fluorescence and infrared spectroscopies will yield identical folding times (so-called two-state folding). We believe that the D14A mutant will violate this hypothesis, while the other two will conform, based on our design.