Fluorescence and Thermodynamics Studies with Proteins

9407167 Eftink在上一个资助期内，我们开发了一种多维分光光度计，它可以同时自动测量多种类型的光学数据（圆二色性，荧光，吸光度和光散射），这些数据是温度，添加变性剂或静水压力的函数。 建议通过增加全光谱荧光检测（通过增强二极管阵列）和通过在滴定实验期间增加自动pH（或特定离子）测量的能力来进一步开发该方法。 此外，我们将为该仪器添加一个停止流动附件，以实现对大分子折叠/展开时间过程的多维监测。 建议通过系统地扰动蛋白质的方式，预计将导致可观察到的人口的折叠中间体，重点是新的，多维光谱方法的持续发展，以扩展我们的研究的热力学蛋白质折叠。 我们将使用这些多维方法来研究所选蛋白质的解折叠，选择这些蛋白质是因为a）它们代表不同折叠基序的例子（即，α螺旋束、β折叠结构、八链α/β桶、具有亚结构域的混合α/β结构、同二聚体蛋白），并且因为B）它们在某些条件下显示出多相转变的证据。 我们的目的是表征结构域/亚基之间的耦合的自由能，并了解影响结构域/亚基之间的相互作用的扰动类型。 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/解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. ***