Fluorescence and Thermodynamics Studies with Proteins

9407167 Eftink在上一次授权期内，我们开发了一种多维分光光度计，可以同时自动测量多种类型的光学数据(圆二色、荧光、吸光度和光散射)，作为温度、添加的变性剂或流体静压的函数。建议通过增加全光谱荧光检测(通过增强型二极管阵列)和在滴定实验中增加自动测量pH(或特定离子)的能力来进一步发展该方法。此外，我们将在这台仪器上增加一个停流附件，以实现对大分子折叠/展开的时间过程的多维监控。%建议通过系统地干扰蛋白质来扩展我们对蛋白质折叠热力学的研究，以期导致可观察到的折叠中间体群，并强调继续开发新的多维光谱方法。我们将使用这些多维方法来研究所选蛋白质的展开，这是因为a)它们代表了不同折叠基序的例子(即，α螺旋束、β折叠结构、8链α/β桶、具有亚结构域的混合α/β结构、同源二聚体蛋白质)，以及b)它们在某些条件下显示了多相转变的证据。我们的目的是表征结构域/亚单位之间耦合的自由能，并了解影响结构域/亚单位之间相互作用的微扰类型。第二个目标是继续开发我们的多维分光光度计和相关方法，以扩展我们以多维方式获得蛋白质折叠/去折叠的平衡和动力学数据的能力。***

9407167 Eftink In the last grant period we developed a multi-dimensional spectrophotometer, which enables the simultaneous and automated measurement of multiple types of optical data (circular dichroism, fluorescence, absorbance and light scattering) as a function of temperature, added denaturant, or hydrostatic pressure. It is proposed to further develop this method by adding full spectral fluorescence detection (via an intensified diode array) and by adding the capability of automatic pH (or specific ion) measurement during titration experiments. Additionally we will add a stopped flow accessory to this instrument to enable multi-dimensional monitoring of the time course of the folding/unfolding of macromolecules. %%% It is proposed to extend our studies of the thermodynamics of protein folding by systematically perturbing proteins in ways that are expected to lead to observable populations of folding intermediates, with emphasis on the continued development of new, multi-dimensional spectroscopic methods. We will use these multi-dimensional methods to study the unfolding of selected proteins, which are selected because a) they represent examples of different folding motifs (i.e., alpha helix bundle, beta sheet structure, eight stranded alpha/beta barrel, mixed alpha/beta structures having sub-domains, homodimeric proteins) and because b) they show evidence for a multi-phasic transition under certain conditions. Our intent is to characterize the free energy of coupling between domains/subunits and to appreciate the type of perturbations that affect the interactions between domains/subunits. A second goal is to continue to develop our multi-dimensional spectrophotometer and related methods, to extend our ability to obtain equilibrium and kinetics data for the folding/unfolding of proteins in a multi-dimensional manner. ***