利用核磁共振觀測激發(fā)態(tài)的贗接觸位移
發(fā)布時間：2019-06-20 15:16
【摘要】：此論文包含兩部分內(nèi)容：前三章闡述利用化學(xué)交換飽和轉(zhuǎn)移(chemical exchange saturation transfer, CEST)實驗獲取蛋白質(zhì)激發(fā)態(tài)贗接觸位移(pseudocontact shifts, PCSs)的研究；第四章闡述利用核磁共振(nuclear magnetic resonance, NMR)對蛋白質(zhì)-配體弱相互作用的初步研究。蛋白質(zhì)的激發(fā)態(tài)構(gòu)象在蛋白折疊、分子識別、酶催化等過程中起重要作用。過去幾十年中,晶體學(xué)和核磁共振在解析蛋白質(zhì)基態(tài)結(jié)構(gòu)方面成果頗豐,但是激發(fā)態(tài)由于布居數(shù)低(5%),對大多數(shù)實驗都處于“不可見”狀態(tài)。弛豫擴散(relaxation dispersion)等核磁實驗可以檢測蛋白質(zhì)在微秒到毫秒時間尺度的運動。最近發(fā)展的CEST實驗可以研究更慢的時間尺度(～2到100毫秒),并獲得激發(fā)態(tài)的化學(xué)位移。然而僅靠化學(xué)位移來搭建激發(fā)態(tài)的原子結(jié)構(gòu)模型仍然是一個很大的挑戰(zhàn),因此亟需發(fā)展富含結(jié)構(gòu)信息的新技術(shù)來描述具有重要功能的激發(fā)態(tài)構(gòu)象。PCS由于含有長程的距離和方向信息,原則上也可以用于描述蛋白質(zhì)激發(fā)態(tài)的構(gòu)象。在本文中,我們提出一種名為PCS-CEST的方法來觀測低豐度激發(fā)態(tài)的PCS信息�？梢允褂靡痪S或者二維的CEST實驗來測量激發(fā)態(tài)的PCS。我們首先在Abplp SH3-Arklp小肽慢交換體系上驗證了這一方法,其激發(fā)態(tài)的PCS與小肽飽和時的PCS數(shù)據(jù)吻合。然后將其用于研究存在少量折疊過渡態(tài)的HYPA/FBP11 FF結(jié)構(gòu)域,其激發(fā)態(tài)的PCS顯著小于基態(tài)的PCS,說明其低豐度激發(fā)態(tài)構(gòu)象系綜存在部分無規(guī)結(jié)構(gòu)。最后我們提出一種選擇性激發(fā)的一維CEST實驗,可以用更短的時間對激發(fā)態(tài)化學(xué)位移進行更精細的掃描,從而提高數(shù)據(jù)的分辨率。因此PCS-CEST可以有效地獲取慢交換情形下的蛋白質(zhì)激發(fā)態(tài)結(jié)構(gòu)信息。第四章闡述利用核磁共振研究蛋白質(zhì)-配體的弱相互作用。在過去二十年中,基于片段的先導(dǎo)化合物發(fā)現(xiàn)被證明是成果顯著的藥物發(fā)現(xiàn)手段。但由于初始篩選出的小分子親和力較低,獲得復(fù)合物晶體往往會遇到困難。通過核磁共振方法獲取結(jié)合狀態(tài)的少量約束,對于小分子結(jié)合模式的判斷很有幫助,因此NMR是晶體學(xué)以外很好的補充手段。我們首先對LARG PDZ結(jié)構(gòu)域進行了片段篩選(fragment-based screening, FBS),然后嘗試了諸如殘留偶極耦合(residual dipolarcouplings, RDC)、順磁弛豫增強(paramagnetic relaxation enhancement, PRE)以及PCS等多種核磁方法來產(chǎn)生蛋白質(zhì)-小分子的結(jié)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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q61;O482.532
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q61;O482.532
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