蛋白质顺磁标记研究
摘要
顺磁标记是指将含有未成对电子的顺磁性物质引入到蛋白质中得到顺磁限制的方法,常用的顺磁效应主要包括：赝接触位移(PCS)、顺磁弛豫增强(PRE)、残余偶极耦合(RDC)。这些顺磁效应包含丰富的蛋白质结构信息可用来研究蛋白质。由于大多数蛋白质都没有顺磁金属离子的结合位点,所以研究中通常采用不同的方法将金属结合标签连接到蛋白质上,常用的方法有化学标签的二硫键连接和多肽融合。由于二硫键产物在还原环境或高pH条件下不能稳定存在,从而限制了其应用。多肽融合虽然能避免二硫键连接中存在的问题,但是在蛋白质中缺少合适的嵌入位点。因此,本论文探索不同的顺磁标记方式,进而获得蛋白质的顺磁结构限制,研究内容主要包括以下两部分：
一、通过稀土配合物与蛋白质的特异性非共价作用标记蛋白质
在吡啶-2,6-二甲酸(L1)分子上引入不同的取代基得到了一系列衍生物L(L=L2,L3,L4,L5),这些配体都能与稀土形成3：1稳定的配合物[Ln(L)3]n-(L=L1,L2,L3,L4,L5)。实验表明这一系列稀土配合物均能与蛋白质ubiquitin产生特异性的相互作用。通过15N-HSQC实验测定得到了配合物[Ln(L)3]n和ubiquitin相互作用的解离常数Kd,赝接触位移PCS,进而拟合得到不同配合物[Ln(L)3]n-的△χ张量及相关参数。通过分析数据,得到如下结论：
(1)改变配合物[Ln(L)3]n取代基和电荷,可以改变配合物与蛋白质的结合常数。
(2)PCS的大小与解离常数Kd及配合物[Ln(L)3]n-的结构有关。
(3)不同配合物[Ln(L)3]n-可获得不同的△χ张量,快速得到多个角度和距离的限制,可以应用到蛋白质的结构。
(4)由于五种配合物[Ln(L)3]n-的体积大小不同以及ubiquitin相互作用界面的柔性,拟合得到的这五种配合物[Ln(L)3]n的金属离子位置略有不同。
二、通过蛋白质半胱氨酸的巯基与碳碳双键类似Michael加成反应标记蛋白质
在Michael加成反应标记蛋白质的基础上,探索了影响Michael加成反应的因素；研究了经标签修饰的蛋白质与顺磁金属离子的作用。具体实验内容和分析结果如下：
通过1H NMR检测室温下不同pH时标签L6与半胱氨酸Cys和赖氨酸Lys的反应活性,当pH=7.0,7.5,8.0,8.5时,标签L6与Cys都完全反应；在pH=7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5的条件下,标签L6与Lys都不反应。通过上述的实验说明标签L6只与Cys的巯基发生特异性的加成反应。
在室温,pH=7.6的条件下,通过15N-HSQC谱图检测了ubiquitin的4种突变体ubiquitin T22C, ubiquitin A28C, ubiquitin G47C, ubiquitin E64C与标签L6的加成反应,通过比较4种突变体的反应产率来确定它们的反应活性。实验表明蛋白质的4种突变体显示出不同的活性,活性大小顺序为：ubiquitin A28C 通过Michae1加成反应,分别将标签L7,L8共价连接到ubiquitin突变体T22C,A28C上,得到4种相应的复合物ubiquitin T22C-L7, ubiquitin A28C-L7, ubiquitin T22C-L8, ubiquitin A28C-L8。在这4种复合物中加入不同顺磁金属离子Ln3+(Ln3+=Dy3+, Tb3+, Tm3+, Yb3+),以抗磁金属离子y3+作为参比,分别计算4种复合物中加入不同顺磁金属离子时的PCS值。进而,利用复合物ubiquitinT22C-L7中滴加顺磁金属离子Tm3+和Yb3+的PCS数据来拟合出AZ张量及相关参数,由于残基22位于ubiquitin结构较柔性的loop区域,loop区的运动导致PCS平均化,产生较小的△X张量。
A paramagnetic center in a protein leads to the interaction of the unpaired electron with the nuclear spins of the protein. This results in several paramagnetic effects such as pseudocontact shifts (PCS), paramagnetic relaxation enhancement (PRE), residual dipolar couplings (RDC). These effects provide long-range distance and angular information for proteins, which are valuable in the structure determination of proteins. Because most proteins are diamagnetic and usually don't have paramagnetic metal-binding motifs, the method of site-specific labeling of proteins with lanthanide-binding ligands have been introduced, including attaching a chemically synthetized ligand through the disulfide bond, insertion of a lanthanide-binding peptide into a protein. However, the instability of the disulfide-bond tether precludes the subsequent use of the method in the presence of reducing agents or at high pH conditions. Although the insertion of a lanthanide binding peptide into the loop of a protein would avoid this complication, there are only a few suitable fusion sites in a protein if any. Herein, we concentrate on the different technical methods obtaining a set of structural restraints through site-specific labeling of proteins. The thesis includes the following two parts:
1. Noncovalent tagging proteins with paramagnetic lanthanide complexes
Starting from pyridine-2,6-dicarboxylic acid (L1), a series of novel pyridine-2,6-dicarboxylic acid derivatives(L=L2,L3,L4,L5) were synthesized by introducing the different substitution patterns.Lanthanide complexes [Ln(L)3]n-(L=L1,L2,L3,1.4,1.5) were formed by paramagnetic lanthanide ions and the different DPA derivatives. The following experiments showed that [Ln(L)3]n-complexes could interact site-specifically with ubiquitin. The binding affinity of [Ln(L)3]n-complexes with ubiquitin and PCSs can be measured by15N-HSQC spectra. The Δx, tensor parameters were calculated by the simulation of PCS data. The following conclusions were obtained through data analysis:
(1)The binding affinity between the protein and [Ln(L)3]n" complexes could be adjusted by changing either the charge or the substitution pattern on the organic ligand.
(2)PCS not only depended on the binding affinity of the metal complex towards the protein but also on the geometry of the metal complex.
(3)[Ln(L)3]n-complexes offered diverse paramagnetic Δx tensors and quickly obtained multiple angles and distance restraints, which were valuable for the structural determination of the proteins.
(4)The determined metal positions of the lanthanide complexes of the different DPA derivatives were not well-converged into one fixed position, which was probably due to the different sizes of the [Ln(L)3]n-complexes and also to the plasticity of the ubiquitin-binding interface.
2. Site-specific labeling of proteins through Michael addition reaction between C-C double bonds and cysteine of protein
On the basis of Michael addition reaction, several factors affecting the Michael addition reaction were explored. In addition, the interactions between labeling proteins and paramagnetic lanthanide ions had been studied. With the studying of influencing factor and interaction, the experiment contents and the results were as follow:
The reaction of tag (Le) with cysteine and lysine were monitored by1H NMR at room temperature. The cysteine reacted fast with L6in the pH range of7.0to8.5. The reaction of L6and Lysine was studied in the pH range of7.0to10.5.1H NMR spectra showed that lysine did not react with L6. Based on the above-mentioned results, we concludeded that the reaction of L6and cysteine was specific for the sulfhydryl group.
The reactions of four T22C, A28C, G47C and E64C mutants of human ubiquitin with L6were monitored by15N-HSQC spectra at room temperature and at pH7.6.The reactive activities were determined by comparison the ligation yield under the same reaction condition.The four mutants showed different reaction activities following ubiquitin A28C The ubiquitin T22C-L7, ubiquitin A28C-L7, ubiquitin T22C-L8, ubiquitin A28C-L8adducts were obtained through the addition reactions between two T22C, A28C mutants of ubiquitin and L7, L8.15N-HSQC spectra were recorded from the four protein complexes with Dy3+, Tb3+, Tm3+, Yb3+. The PCSs were measured in the four protein complexes with paramagnetic lanthanides by using the complex with Y3+as the diamagnetic reference. In addition, the PCS measured for the ubiquitin T22C-L7complexes with Tm3+, Yb3+were used to determine Δχ tensors parameters. Because T22are located in a flexible part of ubiquitin, the mobility of the loop averages the size of PCS, resulting in smaller magnitude of Δχ tensors.
一、通过稀土配合物与蛋白质的特异性非共价作用标记蛋白质
在吡啶-2,6-二甲酸(L1)分子上引入不同的取代基得到了一系列衍生物L(L=L2,L3,L4,L5),这些配体都能与稀土形成3：1稳定的配合物[Ln(L)3]n-(L=L1,L2,L3,L4,L5)。实验表明这一系列稀土配合物均能与蛋白质ubiquitin产生特异性的相互作用。通过15N-HSQC实验测定得到了配合物[Ln(L)3]n和ubiquitin相互作用的解离常数Kd,赝接触位移PCS,进而拟合得到不同配合物[Ln(L)3]n-的△χ张量及相关参数。通过分析数据,得到如下结论：
(1)改变配合物[Ln(L)3]n取代基和电荷,可以改变配合物与蛋白质的结合常数。
(2)PCS的大小与解离常数Kd及配合物[Ln(L)3]n-的结构有关。
(3)不同配合物[Ln(L)3]n-可获得不同的△χ张量,快速得到多个角度和距离的限制,可以应用到蛋白质的结构。
(4)由于五种配合物[Ln(L)3]n-的体积大小不同以及ubiquitin相互作用界面的柔性,拟合得到的这五种配合物[Ln(L)3]n的金属离子位置略有不同。
二、通过蛋白质半胱氨酸的巯基与碳碳双键类似Michael加成反应标记蛋白质
在Michael加成反应标记蛋白质的基础上,探索了影响Michael加成反应的因素；研究了经标签修饰的蛋白质与顺磁金属离子的作用。具体实验内容和分析结果如下：
通过1H NMR检测室温下不同pH时标签L6与半胱氨酸Cys和赖氨酸Lys的反应活性,当pH=7.0,7.5,8.0,8.5时,标签L6与Cys都完全反应；在pH=7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5的条件下,标签L6与Lys都不反应。通过上述的实验说明标签L6只与Cys的巯基发生特异性的加成反应。
在室温,pH=7.6的条件下,通过15N-HSQC谱图检测了ubiquitin的4种突变体ubiquitin T22C, ubiquitin A28C, ubiquitin G47C, ubiquitin E64C与标签L6的加成反应,通过比较4种突变体的反应产率来确定它们的反应活性。实验表明蛋白质的4种突变体显示出不同的活性,活性大小顺序为：ubiquitin A28C
A paramagnetic center in a protein leads to the interaction of the unpaired electron with the nuclear spins of the protein. This results in several paramagnetic effects such as pseudocontact shifts (PCS), paramagnetic relaxation enhancement (PRE), residual dipolar couplings (RDC). These effects provide long-range distance and angular information for proteins, which are valuable in the structure determination of proteins. Because most proteins are diamagnetic and usually don't have paramagnetic metal-binding motifs, the method of site-specific labeling of proteins with lanthanide-binding ligands have been introduced, including attaching a chemically synthetized ligand through the disulfide bond, insertion of a lanthanide-binding peptide into a protein. However, the instability of the disulfide-bond tether precludes the subsequent use of the method in the presence of reducing agents or at high pH conditions. Although the insertion of a lanthanide binding peptide into the loop of a protein would avoid this complication, there are only a few suitable fusion sites in a protein if any. Herein, we concentrate on the different technical methods obtaining a set of structural restraints through site-specific labeling of proteins. The thesis includes the following two parts:
1. Noncovalent tagging proteins with paramagnetic lanthanide complexes
Starting from pyridine-2,6-dicarboxylic acid (L1), a series of novel pyridine-2,6-dicarboxylic acid derivatives(L=L2,L3,L4,L5) were synthesized by introducing the different substitution patterns.Lanthanide complexes [Ln(L)3]n-(L=L1,L2,L3,1.4,1.5) were formed by paramagnetic lanthanide ions and the different DPA derivatives. The following experiments showed that [Ln(L)3]n-complexes could interact site-specifically with ubiquitin. The binding affinity of [Ln(L)3]n-complexes with ubiquitin and PCSs can be measured by15N-HSQC spectra. The Δx, tensor parameters were calculated by the simulation of PCS data. The following conclusions were obtained through data analysis:
(1)The binding affinity between the protein and [Ln(L)3]n" complexes could be adjusted by changing either the charge or the substitution pattern on the organic ligand.
(2)PCS not only depended on the binding affinity of the metal complex towards the protein but also on the geometry of the metal complex.
(3)[Ln(L)3]n-complexes offered diverse paramagnetic Δx tensors and quickly obtained multiple angles and distance restraints, which were valuable for the structural determination of the proteins.
(4)The determined metal positions of the lanthanide complexes of the different DPA derivatives were not well-converged into one fixed position, which was probably due to the different sizes of the [Ln(L)3]n-complexes and also to the plasticity of the ubiquitin-binding interface.
2. Site-specific labeling of proteins through Michael addition reaction between C-C double bonds and cysteine of protein
On the basis of Michael addition reaction, several factors affecting the Michael addition reaction were explored. In addition, the interactions between labeling proteins and paramagnetic lanthanide ions had been studied. With the studying of influencing factor and interaction, the experiment contents and the results were as follow:
The reaction of tag (Le) with cysteine and lysine were monitored by1H NMR at room temperature. The cysteine reacted fast with L6in the pH range of7.0to8.5. The reaction of L6and Lysine was studied in the pH range of7.0to10.5.1H NMR spectra showed that lysine did not react with L6. Based on the above-mentioned results, we concludeded that the reaction of L6and cysteine was specific for the sulfhydryl group.
The reactions of four T22C, A28C, G47C and E64C mutants of human ubiquitin with L6were monitored by15N-HSQC spectra at room temperature and at pH7.6.The reactive activities were determined by comparison the ligation yield under the same reaction condition.The four mutants showed different reaction activities following ubiquitin A28C
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