Nmr Studies Of Biomolecular Structure, Function, And Dynamics

生物分子结构、功能和动力学的核磁共振研究

• 批准号: 7734459
• 负责人: Robert E London
• 金额: $ 87.97万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734459
• 关键词: Active Sites; Address; Allergens; Allergic; Amides; Amino Acids; Antibiotics; Area; Arts; Asthma; Autoimmune Diseases; Bacteria; Behavior; Binding; Biological; Biological Factors; Breathing; C-terminal; Carbon; Chemicals; Code; Complex; Conflict (Psychology); Crystallization; DNA; DNA Damage; DNA Repair; Data; Databases; Dependence; Dermatophagoides pteronyssinus antigen p 5; Dermatophagoides pteronyssinus antigen p 7; Development; Dihydrofolate Reductase; Discrimination; Disease; Environmental Health; Enzymes; Evolution; Exhibits; Exposure to; Folic Acid Antagonists; Genetic; Genomic Instability; Genomics; Genotoxic Stress; Glutamine; Goals; Health; Helix-Turn-Helix Motifs; Homology Modeling; House Dust Mite Allergens; Human; Hydrogen Bonding; Hypersensitivity; Indium; Label; Lead; Lyase; Mammals; Methionine; Methodology; Modeling; Modification; Molecular; Molecular Conformation; Mutation; NADP; NMR Spectroscopy; Nature; Niacinamide; Nucleotide Excision Repair; Numbers; Oxidation-Reduction; Pharmaceutical Preparations; Phase; Plasmids; Positioning Attribute; Probability; Prokaryotic Cells; Property; Protein Overexpression; Proteins; Protons; Pteridines; Range; Reaction; Relative (related person); Research; Research Design; Research Project Grants; Resolution; Site-Directed Mutagenesis; Solutions; Stress; Structure; Substrate Specificity; Surface; Surveys; System; Testing; Time; Trimethoprim; Vertebral column; Work; arginyllysine; bacterial resistance; base; cofactor; dihydrofolate; dihydrofolate reductase type II; dimer; expectation; gene correction; improved; insight; interest; isoleucylvaline; leucine methyl ester; leucylmethionine; lysylglutamic acid; macromolecule; pol genes; pyridine nucleotide; pyroglyphid; repaired; restraint; vector

This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the area of environmental health. The primary emphasis during the recent review period involves several different research projects: 1) characterization of the structural and dynamic behavior of the bacterial nucleotide excision repair protein UvrB in order to understand the molecular basis for damage recognition; 2) fundamental studies of the relationships between the 13C shifts of amino acids and their conformation in proteins; 3) structural work on dust mite allergens; and 4) structural analysis of type II Dihydrofolate reductase (DHFR), a plasmid-encoded enzyme that confers resistance to bacteria-targeted antifolate antibiotics. Project 1. Nucleotide excision repair (NER) is an essential DNA repair mechanism which is highly conserved among all biological systems. Of all the repair mechanisms, it exhibits the greatest versatility, characterized by the extremely heterogeneous types of damage shown to be recognized and corrected. In prokaryotes, NER is accomplished by three enzymes: Uvr A, B and C. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the solution behavior of UvrB, we have performed NMR studies on methyl-13C-methionine-labeled UvrB (MW = 75 kD) from B. caldotenax. The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Analysis of the resonance assigned to M632, located at the interface of the domain 4 dimer, demonstrated that a UvrB-UvrB-domain 4 heterodimer could form, but that a UvrB-UvrB homodimer appeared to be disfavored in solution. In order to characterize the nature of the interaction between UvrB and DNA, subsequent studies have investigated the interaction of UvrB with DNA hairpins. Based on crystal structure data showing the involvement of the UvrB hairpin (residues S91-D117), we have introduced methionine probe residues at positions 109 and 115 using site-directed mutagenesis. Initial results demonstrate that the spectral changes observed in the presence of hairpin DNA are consistent with expectations based on the crystal structure. In order to extend these conclusions, studies of other modified DNAs are currently in progress. Project 2. Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of sidechain 13C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cγ resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (non-proton) γ-substituents and with gauche conformational orientations of γ-substituents. The 13C shift/conformation correlations are most apparent for Cγ carbons, but also can be observed at positions further from the backbone. Intra-residue steric conflict leads to a correlation between upfield-shifted sidechain 13C resonances and statistically lower probabilities in surveys of protein sidechain conformation. Illustrative applications to the DNA pol lambda lyase domain, and to dihydrofolate reductase are discussed. In the latter case, 13C shift analysis indicates that the conformation of the remote residue V119 on the βF-βG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. Anlaysis of 13C shift data for protein sidechains should provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, δCδ1-δCδ2, on both χ1 and χ2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available. Project 3. Inhalation allergy to house dust mite allergens is among the most prevalent allergic diseases. Our studies of the structure of dust mite allergens have focused in two specific examples: Der p 5 and Der p 7. We found that Der p 5 can be overexpressed with a GST-tag and purified after TEV cleavage. NMR spectra of the protein were of marginal quality, however, the protein crystallized readily and gave data to 3.2 A. Unfortunately, even an NMR based homology model using the structure of Blo t 5 was unable to solve the phase problem. Currently the coding sequence has been moved into vectors containing an MBP tag that has been modified for enhanced crystallization. It is anticipated that co-crystals of MBP-Der p 5 should solve the phase problem. Similarly, various constructs of Der p 7 have been tested, and a construct that starts at residue 18 expresses the most soluble protein. NMR spectra were evaluated as poor. The protein can be concentrated to greater than 50 mg/ml and crystallizes readily. However, again, the crystals diffract from 3-11 A which is insufficient to solve the phase problem without a high resolution homology model. Again our current strategy is to try co-crystals using a modified MBP vector. Project 4. Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We have determined the first structure of a ternary R67 DHFR-dihydrofolate-NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a molecular clamp holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally-challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.

该项目利用最先进的核磁共振波谱来研究环境健康领域持续关注的问题。 最近审查期间的主要重点涉及几个不同的研究项目：1）表征细菌核苷酸切除修复蛋白UvrB的结构和动态行为，以了解损伤识别的分子基础； 2）蛋白质中氨基酸13C位移与其构象关系的基础研究； 3）尘螨过敏原的结构工作； 4) II 型二氢叶酸还原酶 (DHFR) 的结构分析，DHFR 是一种质粒编码的酶，可赋予细菌靶向抗叶酸抗生素的抗性。 项目1. 核苷酸切除修复（NER）是一种重要的DNA修复机制，在所有生物系统中高度保守。 在所有修复机制中，它表现出最大的多功能性，其特点是可以识别和纠正极其异质的损伤类型。 在原核生物中，NER 由三种酶完成：Uvr A、B 和 C。完整 UvrB 晶体结构中 C 端结构域 4 的明显无序限制了结构信息； 在溶液中，发现分离的结构域4形成螺旋-环-螺旋二聚体。为了深入了解 UvrB 的溶液行为，我们对来自 B. caldotenax 的甲基 13C-蛋氨酸标记的 UvrB (MW = 75 kD) 进行了 NMR 研究。 13 个甲​​基共振是根据定点诱变和结构域删除进行分配的。 对位于结构域 4 二聚体界面处的 M632 的共振分析表明，可以形成 UvrB-UvrB-结构域 4 异二聚体，但 UvrB-UvrB 同二聚体似乎不利于溶液。 为了表征 UvrB 与 DNA 之间相互作用的本质，后续研究调查了 UvrB 与 DNA 发夹的相互作用。 基于显示 UvrB 发夹（残基 S91-D117）参与的晶体结构数据，我们使用定点诱变在位置 109 和 115 处引入了蛋氨酸探针残基。 初步结果表明，在发夹 DNA 存在的情况下观察到的光谱变化与基于晶体结构的预期一致。 为了扩展这些结论，目前正在进行其他修饰 DNA 的研究。 项目 2. 将来自 BiomagResDataBank 的化学位移数据和来自蛋白质数据库的构象数据关联起来，以探索侧链 13C 共振位移的构象依赖性。 与基于空间压缩的预测一致，Thr、Val、Ile、Leu、Met、Arg、Lys、Glu 和 Gln 残基的 Cγ 共振的高场位移与重原子（非质子）γ 取代基的数量和具有 γ-取代基的稀疏构象取向。 13C 位移/构象相关性对于 Cγ 碳最为明显，但也可以在远离主链的位置观察到。 残基内空间冲突导致上场移动的侧链 13C 共振与蛋白质侧链构象调查中统计上较低的概率之间存在相关性。 讨论了 DNA pol λ 裂合酶结构域和二氢叶酸还原酶的示例性应用。 在后一种情况下，13C位移分析表明βF-βG环上远程残基V119的构象与结合的吡啶核苷酸辅因子的氧化还原状态相关，为区分底物和产物提供了基础。 蛋白质侧链 13C 位移数据的分析应该为分析大型氘化蛋白质的构象变化提供有用的基础。此外，亮氨酸甲基位移差异 δCδ1-δCδ2 对 χ1 和 χ2 的很大依赖性足以允许该参数在立体特异性分配数据可用的情况下用作结构计算中的限制。 项目 3. 对屋尘螨过敏原的吸入过敏是最常见的过敏性疾病之一。 我们对尘螨过敏原结构的研究集中在两个具体例子：Der p 5 和 Der p 7。我们发现 Der p 5 可以用 GST 标签过表达，并在 TEV 裂解后纯化。 该蛋白质的 NMR 谱质量较差，但该蛋白质很容易结晶并提供 3.2 A 的数据。不幸的是，即使使用 Blo t 5 结构的基于 NMR 的同源模型也无法解决相位问题。 目前，编码序列已被转移到含有 MBP 标签的载体中，该标签已被修改以增强结晶。 预计MBP-Der p 5 的共晶应解决相位问题。 类似地，已经测试了 Der p 7 的各种构建体，从残基 18 开始的构建体表达最可溶的蛋白质。 NMR谱被评价为差。 蛋白质可浓缩至大于 50 mg/ml，并且易于结晶。 然而，晶体再次从 3-11 A 衍射，如果没有高分辨率同源模型，这不足以解决相位问题。 同样，我们当前的策略是尝试使用改良的 MBP 载体进行共晶。 项目 4. II 型二氢叶酸还原酶 (DHFR) 是一种质粒编码的酶，可赋予细菌 DHFR 靶向抗叶酸药物的抗性。它形成具有作为活性位点的中心孔的对称同源四聚体。 其不寻常的结构导致了混杂的结合表面，可容纳二氢叶酸 (DHF) 底物或 NADPH 辅因子，这对了解其底物特异性和反应机制的努力构成了重大限制。 我们已经确定了三元 R67 DHFR-二氢叶酸-NADP+ 催化复合物的第一个结构，解析为 1.26 A。该结构提供了第一个清晰的图片，说明这种缺乏 I 型 DHFR 中普遍存在的活性位点羧基残基的酶如何能够发挥作用。 在催化复合物中，两个对称相关的 I68 残基的极性主链原子提供了与烟酰胺环上的甲酰胺和蝶啶上的 N3-O4 酰胺功能相互作用的识别基序。 这组相互作用将底物和辅因子的芳环定向在相对的内几何形状中，其中反应中心保持紧密接近。 此外，由两对 Y69-Q67-Q67'-Y69' 残基组成的中心氢键网络提供了异常紧密的界面，这似乎充当分子夹，将底物固定在有利于氢化物转移的方向上。 除了提供关于这种极其不寻常的酶如何发挥作用的第一个清晰的见解之外，三元复合物的结构还提供了对突变挑战的酶（即，其进化仅限于四个残基的酶）如何发挥作用的一般见解。一次活性位点突变克服了这一基本限制。

项目成果

NMR assignment of protein side chains using residue-correlated labeling and NOE spectra.

使用残基相关标记和 NOE 谱对蛋白质侧链进行 NMR 分配。

• DOI: 10.1016/j.jmr.2003.08.006
• 发表时间: 2003
• 期刊: Journal of magnetic resonance (San Diego, Calif. : 1997)
• 作者: Mueller,GeoffreyA;Kirby,ThomasW;DeRose,EugeneF;London,RobertE
• 通讯作者: London,RobertE

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups.

• DOI: 10.1007/s10858-010-9436-6
• 发表时间: 2010-09
• 期刊: Journal of biomolecular NMR
• 影响因子: 2.7
• 作者: Butterfoss GL;DeRose EF;Gabel SA;Perera L;Krahn JM;Mueller GA;Zheng X;London RE
• 通讯作者: London RE

Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate.

酪氨酸中的光激活 h/d 交换：自由基阴离子中间体的参与。

• DOI: 10.1021/ja055011c
• 发表时间: 2006
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: London,RobertE;Gabel,ScottA
• 通讯作者: Gabel,ScottA

Characterization of the redox transition of the XRCC1 N-terminal domain.

XRCC1 N 端结构域氧化还原转变的表征。

• DOI: 10.1016/j.str.2014.09.012
• 发表时间: 2014
• 期刊: Structure (London, England : 1993)
• 作者: Gabel,ScottA;Smith,CassandraE;Cuneo,MatthewJ;Mueller,GeoffreyA;Kirby,ThomasW;DeRose,EugeneF;Krahn,JunoM;London,RobertE
• 通讯作者: London,RobertE

Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis.

• DOI: 10.1021/ja802729t
• 发表时间: 2008-08-20
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: London RE;Wingad BD;Mueller GA
• 通讯作者: Mueller GA