Physical Biochemistry Of Macromolecules

大分子物理生物化学

• 批准号: 7593808
• 负责人: Marc Lewis
• 金额: $ 2.64万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 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/7593808
• 关键词: Behavior; Biochemistry; Biological; Cells; Collaborations; Complex; DNA; DNA Maintenance; DNA Polymerase beta; DNA Repair; DNA ligase I; DNA-(apurinic or apyrimidinic site) lyase; Data Analyses; Dependence; Dependency; Entropy; Enzymes; Eukaryotic Cell; Exhibits; Financial compensation; Free Energy; Genetic Transcription; Heating; Histidine; Histones; Individual; Investigation; Journals; Laboratories; Malignant Neoplasms; Malignant neoplasm of lung; Manuscripts; Molecular; Molecular Chaperones; National Institute of Diabetes and Digestive and Kidney Diseases; Nuclear; Oligonucleotides; Peptide Nucleic Acids; Preparation; Proliferating Cell Nuclear Antigen; Property; Proteins; Publishing; Purpose; RTH-1 Nuclease; Research; Role; Specificity; Structure; TP53 gene; Techniques; Temperature; Thermodynamics; Transcription Initiation; Value Meaning; Variant; Vertebral column; Work; amyloid formation; analytical ultracentrifugation; base; dimer; enthalpy; interest; macromolecule; mathematical model; monomer; mutant; novel; physical property; stoichiometry

Collaborative studies with the laboratory of Dr. Samuel Wilson (NIEHS) on proteins involved in DNA transcription initiation and in DNA repair have continued. These have included work on the interactions between DNA Ligase I and the replication protein, proliferating cell nuclear antigen (PCNA); the interaction mechanisms between the XRCC1(1-183) protein and DNA polymerase-beta and its subdomains; the interactions between AP endonuclease and DNA, DNA polymerase-beta and DNA, and both together with DNA. Research has been completed on two aspects of the studies on DNA enzymes, the thermodynamics of oligomerization of PCNA and the interaction of PCNA with histidine-tagged and untagged FEN-1. Two manuscripts are in preparation. It is anticipated that this work will continue in the coming year. In a collaborative study with the laboratory of Dr. Carl Wu (NCI), we have demonstrated that the proteins Chz1, Htz1, and H2B form a reversible complex with a 1:1:1 stoichiometry. This complex appears to be a novel histone chaperone that has specificity for H2AZ and can deliver the histone variant for SWR1-dependent histone replacement in eukaryotic cells. A manuscript entitled "Chz1, a Novel Nuclear Chaperone for Histone H2AZ" has been published in the journal "Molecular Cell." In a collaborative study with the laboratory of Dr. Daniel Appella (NIDDK), we have been studying the self-associative properties of a synthetic oligonucleotide (referred to here as DNA) and a similar molecule with the same bases attached to a polypepetide backbone (referred to here as PNA). We have demonstrated that the DNA is extremely thermally stable and undergoes a reversible dimer-tetramer self-association with a mean free energy change of -6.88 kcal/mol that has very little temperature dependence and is virtually totally of enthalpic origin. This is entirely consistent with its structure. We find that the PNA has significantly less stability than the DNA, showing signs of degradation at 34 deg. C. It undergoes a monomer-tetramer self-association with a mean free energy change of -7.7 kcal/mol, a weaker self-association than that exhibited by the DNA, since that is for a dimer-tetramer association and the PNA undergoes a monomer-tetramer association. The PNA self-association exhibits little temperature dependence and also is virtually totally of enthalpic origin, again consistent with its structure. A mixture of the DNA and the PNA associates very strongly with a mean free energy change of -19.6 kcal/mol. However, it exhibits unusual temperature dependency and preliminary analysis gave unexpected values for the thermodynamic parameters. The mean value of the change in enthalpy was -3.4 kcal/mol; the mean value for the change in entropy was 59 cal/mol/K; and the mean value for the specific heat content was -2.5 kcal/mol/K. Thus, the values of the change in free energy appear to be essentially a case of enthalpy-entropy compensation. While this is quite common in protein-protein association, it is quite unexpected here and will require considerable additional studies to explain. This is in marked contrast to the thermodynamic behavior of either the DNA or the PNA. Additionally, at temperatures of 28 deg. C and above, no heterotetramer was detectable. Whether this is due to the initiation of degradation of the PNA or the formation of some structure other than heterotetramer remains to be determined. This problem is of considerable biological interest since the PNAs appear to be a particularly specific means for the inhibition of DNA transcription and may have a significant role in the control of malignancy. The results of a collaboration with the laboratory of Dr. Ettore Appella (NCI), performed several years ago, entitled "Unfolding, Aggregation and Amyloid Formation by the Tetramerization Domain From Mutant p53 Associated With Lung Cancer" has been published in the journal "Biochemistry."

继续与Samuel Wilson博士实验室（NIEHS）合作研究涉及DNA转录起始和DNA修复的蛋白质。 这些研究包括DNA连接酶I与复制蛋白、增殖细胞核抗原（PCNA）之间的相互作用; XRCC 1（1-183）蛋白与DNA聚合酶β及其亚结构域之间的相互作用机制; AP核酸内切酶与DNA、DNA聚合酶β与DNA以及两者与DNA之间的相互作用。 对DNA酶的研究主要集中在两个方面，即PCNA寡聚化的热力学和PCNA与组氨酸标记和未标记的FEN-1的相互作用。 两份手稿正在编写中。 预计这项工作将在明年继续进行。 在与Carl Wu博士（NCI）实验室的一项合作研究中，我们证明了蛋白质Chz 1，Htz 1和H2 B形成了1：1：1化学计量的可逆复合物。 这种复合物似乎是一种新的组蛋白伴侣，对H2 AZ具有特异性，并可以在真核细胞中提供SWR 1依赖的组蛋白替代的组蛋白变体。 一篇题为“Chz 1，a Novel Nuclear Chaperone for Histone H2 AZ”的论文发表在《分子细胞》杂志上。" 在与丹尼尔阿佩拉博士（NIDDK）实验室的合作研究中，我们一直在研究合成寡核苷酸（此处称为DNA）和具有连接到多肽骨架的相同碱基的类似分子（此处称为PNA）的自缔合特性。 我们已经证明，DNA是非常热稳定的，并经历了可逆的二聚体-四聚体自缔合的平均自由能变化为-6.88千卡/摩尔，具有非常小的温度依赖性，几乎完全是双链起源。 这与其结构完全一致。 我们发现PNA的稳定性明显低于DNA，在34 ℃下显示出降解迹象。C. 它经历了单体-四聚体自缔合，平均自由能变化为-7.7 kcal/mol，比DNA表现出的自缔合更弱，因为这是二聚体-四聚体缔合，而PNA经历了单体-四聚体缔合。 PNA自缔合几乎没有温度依赖性，并且几乎完全是焓起源，这再次与其结构一致。 DNA和PNA的混合物与-19.6 kcal/mol的平均自由能变化非常强烈地结合。 然而，它表现出不寻常的温度依赖性和初步的分析给了意想不到的值的热力学参数。 焓变的平均值为-3.4 kcal/mol;熵变的平均值为59 cal/mol/K;比热容的平均值为-2.5 kcal/mol/K。 因此，自由能的变化值似乎基本上是熵-熵补偿的情况。 虽然这在蛋白质-蛋白质缔合中很常见，但在这里却很出乎意料，需要大量的额外研究来解释。 这与DNA或PNA的热力学行为形成鲜明对比。 此外，在28度的温度下，C及以上，未检测到异源四聚体。 这是否是由于PNA降解的起始或异源四聚体以外的一些结构的形成仍有待确定。 这个问题具有相当大的生物学意义，因为PNA似乎是抑制DNA转录的特别特异的手段，并且可能在控制恶性肿瘤中具有重要作用。 与Ettore Appella博士（NCI）的实验室合作的结果，几年前进行，题为“通过与肺癌相关的突变体p53的四聚化结构域展开，聚集和淀粉样蛋白形成”，已发表在“生物化学”杂志上。"