利用核磁共振观测激发态的赝接触位移
发布时间：2019-06-20 15:16
【摘要】：此论文包含两部分内容：前三章阐述利用化学交换饱和转移(chemical exchange saturation transfer, CEST)实验获取蛋白质激发态赝接触位移(pseudocontact shifts, PCSs)的研究；第四章阐述利用核磁共振(nuclear magnetic resonance, NMR)对蛋白质-配体弱相互作用的初步研究。蛋白质的激发态构象在蛋白折叠、分子识别、酶催化等过程中起重要作用。过去几十年中,晶体学和核磁共振在解析蛋白质基态结构方面成果颇丰,但是激发态由于布居数低(5%),对大多数实验都处于“不可见”状态。弛豫扩散(relaxation dispersion)等核磁实验可以检测蛋白质在微秒到毫秒时间尺度的运动。最近发展的CEST实验可以研究更慢的时间尺度(～2到100毫秒),并获得激发态的化学位移。然而仅靠化学位移来搭建激发态的原子结构模型仍然是一个很大的挑战,因此亟需发展富含结构信息的新技术来描述具有重要功能的激发态构象。PCS由于含有长程的距离和方向信息,原则上也可以用于描述蛋白质激发态的构象。在本文中,我们提出一种名为PCS-CEST的方法来观测低丰度激发态的PCS信息。可以使用一维或者二维的CEST实验来测量激发态的PCS。我们首先在Abplp SH3-Arklp小肽慢交换体系上验证了这一方法,其激发态的PCS与小肽饱和时的PCS数据吻合。然后将其用于研究存在少量折叠过渡态的HYPA/FBP11 FF结构域,其激发态的PCS显著小于基态的PCS,说明其低丰度激发态构象系综存在部分无规结构。最后我们提出一种选择性激发的一维CEST实验,可以用更短的时间对激发态化学位移进行更精细的扫描,从而提高数据的分辨率。因此PCS-CEST可以有效地获取慢交换情形下的蛋白质激发态结构信息。第四章阐述利用核磁共振研究蛋白质-配体的弱相互作用。在过去二十年中,基于片段的先导化合物发现被证明是成果显著的药物发现手段。但由于初始筛选出的小分子亲和力较低,获得复合物晶体往往会遇到困难。通过核磁共振方法获取结合状态的少量约束,对于小分子结合模式的判断很有帮助,因此NMR是晶体学以外很好的补充手段。我们首先对LARG PDZ结构域进行了片段筛选(fragment-based screening, FBS),然后尝试了诸如残留偶极耦合(residual dipolarcouplings, RDC)、顺磁弛豫增强(paramagnetic relaxation enhancement, PRE)以及PCS等多种核磁方法来产生蛋白质-小分子的结合模型。
[Abstract]:This paper consists of two parts: the first three chapters describe the study of obtaining protein excited pseudo-contact shift (pseudocontact shifts, PCSs) by chemical exchange saturation transfer (chemical exchange saturation transfer, CEST) experiment, and the fourth chapter describes the preliminary study of protein-ligand weak interaction by nuclear magnetic resonance (nuclear magnetic resonance, NMR). The excited state conformations of proteins play an important role in protein folding, molecular recognition, enzyme catalysis and so on. In the past few decades, crystallography and nuclear magnetic resonance (NMR) have achieved a lot of results in analyzing the ground state structure of proteins, but the excited state is in an "invisible" state because of its low population (5%). Relaxation diffusion (relaxation dispersion) and other nuclear magnetic experiments can detect the movement of proteins in the time scale of microseconds to milliseconds. The recently developed CEST experiment can study slower time scales (~ 2 to 100 milliseconds) and obtain the chemical shifts of excited states. However, it is still a great challenge to build the atomic structure model of excited states only by chemical shifts, so it is urgent to develop new techniques rich in structural information to describe excited state conformations with important functions. PCS can also be used in principle to describe the configuration of protein excited states because it contains long-range distance and direction information. In this paper, we propose a method called PCS-CEST to observe the PCS information of low abundance excited states. One-dimensional or two-dimensional CEST experiments can be used to measure the PCS. of excited states. We first verified this method on the Abplp SH3-Arklp small peptide slow exchange system, and the excited PCS coincides with the PCS data when the small peptide is saturated. Then it is used to study the HYPA/FBP11 FF domain with a small amount of folded transition states. The PCS of the excited state is significantly smaller than that of the ground state PCS, indicating that the conformational system of the low abundance excited state exists in some random structures. Finally, we propose a one-dimensional CEST experiment of selective excitation, which can scan the chemical shifts of excited states more precisely in a shorter time, so as to improve the resolution of the data. Therefore, PCS-CEST can effectively obtain the excited state structure information of proteins in the case of slow exchange. In chapter 4, the weak interaction between protein and ligand is studied by nuclear magnetic resonance (NMR). Over the past two decades, fragment-based lead compound discovery has proved to be a promising drug discovery tool. However, due to the low affinity of the small molecules screened initially, it is often difficult to obtain the complex crystals. It is very helpful to judge the binding mode of small molecules by obtaining a small amount of constraints on the binding state by nuclear magnetic resonance (NMR), so NMR is a good complementary method besides crystallography. We first screened the LARG PDZ domain (fragment-based screening, FBS),) and then tried a variety of nuclear magnetic methods, such as residual dipolar coupled (residual dipolarcouplings, RDC), paramagnetically relaxation enhanced (paramagnetic relaxation enhancement, PRE) and PCS, to produce protein-small molecules binding model.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q61;O482.532
本文编号：2503321
[Abstract]:This paper consists of two parts: the first three chapters describe the study of obtaining protein excited pseudo-contact shift (pseudocontact shifts, PCSs) by chemical exchange saturation transfer (chemical exchange saturation transfer, CEST) experiment, and the fourth chapter describes the preliminary study of protein-ligand weak interaction by nuclear magnetic resonance (nuclear magnetic resonance, NMR). The excited state conformations of proteins play an important role in protein folding, molecular recognition, enzyme catalysis and so on. In the past few decades, crystallography and nuclear magnetic resonance (NMR) have achieved a lot of results in analyzing the ground state structure of proteins, but the excited state is in an "invisible" state because of its low population (5%). Relaxation diffusion (relaxation dispersion) and other nuclear magnetic experiments can detect the movement of proteins in the time scale of microseconds to milliseconds. The recently developed CEST experiment can study slower time scales (~ 2 to 100 milliseconds) and obtain the chemical shifts of excited states. However, it is still a great challenge to build the atomic structure model of excited states only by chemical shifts, so it is urgent to develop new techniques rich in structural information to describe excited state conformations with important functions. PCS can also be used in principle to describe the configuration of protein excited states because it contains long-range distance and direction information. In this paper, we propose a method called PCS-CEST to observe the PCS information of low abundance excited states. One-dimensional or two-dimensional CEST experiments can be used to measure the PCS. of excited states. We first verified this method on the Abplp SH3-Arklp small peptide slow exchange system, and the excited PCS coincides with the PCS data when the small peptide is saturated. Then it is used to study the HYPA/FBP11 FF domain with a small amount of folded transition states. The PCS of the excited state is significantly smaller than that of the ground state PCS, indicating that the conformational system of the low abundance excited state exists in some random structures. Finally, we propose a one-dimensional CEST experiment of selective excitation, which can scan the chemical shifts of excited states more precisely in a shorter time, so as to improve the resolution of the data. Therefore, PCS-CEST can effectively obtain the excited state structure information of proteins in the case of slow exchange. In chapter 4, the weak interaction between protein and ligand is studied by nuclear magnetic resonance (NMR). Over the past two decades, fragment-based lead compound discovery has proved to be a promising drug discovery tool. However, due to the low affinity of the small molecules screened initially, it is often difficult to obtain the complex crystals. It is very helpful to judge the binding mode of small molecules by obtaining a small amount of constraints on the binding state by nuclear magnetic resonance (NMR), so NMR is a good complementary method besides crystallography. We first screened the LARG PDZ domain (fragment-based screening, FBS),) and then tried a variety of nuclear magnetic methods, such as residual dipolar coupled (residual dipolarcouplings, RDC), paramagnetically relaxation enhanced (paramagnetic relaxation enhancement, PRE) and PCS, to produce protein-small molecules binding model.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q61;O482.532
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