Single-molecule fluorescence studies of protein folding

描述(由申请人提供)：了解蛋白质折叠和解折叠的途径，并绘制部分折叠的中间状态图，对于我们破译错误折叠疾病的生理学至关重要。在蛋白质的能量景观上生成折叠路径图是我们研究的长期目标。在这里，我们建议通过应用强大的单分子荧光方法来研究选定蛋白质系统的折叠来解决这一目标。我们将使用荧光共振能量转移(FRET)来跟踪捕获在表面束缚的脂泡中的蛋白质分子的构象动力学，这是我们实验室开发的一种独特的方法。囊泡将在分子具有相等折叠和解折叠速率的条件下制备，因此单分子轨迹将显示包括部分折叠的中间体在内的构象状态之间的频繁转变。对轨迹的分析将产生关于折叠路径和折叠中间体之间相互转化速率的丰富信息。此外，对自由扩散分子的研究将探索蛋白质在折叠过程中的整体结构变化。我们将首先研究由62个氨基酸组成的蛋白质L，它是一个从整体实验中得知的两态文件夹。我们将在单分子水平上验证这一观察结果，并获得关于蛋白质变性状态的结构信息。我们还将研究一种更大的蛋白质，腺苷酸激酶，正如我们已经展示的那样，它具有非常不同的折叠能量。在蛋白质折叠过程中观察到的中间产物将被仔细绘制出来。这些中间体的低分辨率结构将通过测量几对标记位点来获得。在与耶鲁大学的Lynne Regan的大力合作中，我们将研究四肽重复蛋白的单分子折叠。这个由34个氨基酸重复单元组成的模块化蛋白质家族被认为遵循一种新的折叠范式，该范式基于经典的Ising模型，该模型预测在过渡中点附近有大量的部分折叠状态。单分子测量将探索这一耐人寻味的断言。这项研究将对我们对阿尔茨海默氏症和II型糖尿病等错误折叠疾病的理解产生影响。我们的实验应该帮助我们识别在折叠过程中充当“弱点”的中间结构，导致导致疾病的蛋白质聚集体。

DESCRIPTION (provided by applicant): Understanding pathways of protein folding and unfolding, and mapping partially folded intermediate states, are of utmost importance for our ability to decipher the physiology of misfolding diseases. The generation of maps of folding pathways on the energy landscapes of proteins is the long range goal of our research. Here we propose to address this goal by applying powerful single-molecule fluorescence methodology to study the folding of selected protein systems. We will use fluorescence resonance energy transfer (FRET) to follow conformational dynamics of protein molecules trapped within surface-tethered lipid vesicles, a unique method developed in our lab. Vesicles will be prepared under conditions where molecules have equal folding and unfolding rates, so that single- molecule trajectories will show frequent transitions between conformational states, including partially-folded intermediates. Analysis of trajectories will yield a wealth of information on folding pathways and rates of interconversion between folding intermediates. In addition, studies on freely-diffusing molecules will probe global structural changes of the proteins during folding. We will first study the 62 amino-acid protein L, known to be a two-state folder from bulk experiments. We will verify this observation on the single molecule level, as well as obtain structural information on the denatured state of the protein. We will also study a larger protein, adenylate kinase, which, as we already showed, possesses a very heterogeneous folding energy lansdcape. Intermediates observed during folding of this protein will be carefully mapped. A low-resolution structure of these intermdiates will be obtained by measuring several pairs of labeling sites. In a strong collaboration with Lynne Regan from Yale we will study single-molecule folding of the tetratricopeptide repeat proteins. This family of modular proteins, constructed of 34 amino-acid repeat units, has been suggested to obey a new folding paradigm, based on the classical Ising model, which predicts a large population of partially folded states near the transition midpoint. Single-molecule measurements will probe this intriguing assertion. This study will have impact on our understanding of misfolding diseases, like Alzheimer's and type II diabetes. Our experiments should help us identifying intermediate structures that act as 'weak points' during folding, leading to protein aggregates which are responsible for disease.