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Structural Studies of Large Dynamic Complexes

大型动态复合体的结构研究

基本信息

批准号：
10621354
负责人：
HELEN JANE DYSON
金额：
$ 51.47万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10621354
关键词：
Acquired Immunodeficiency Syndrome Apoptotic Arthritis Asthma Binding Cell Extracts Cell Nucleus Cell physiology Cell-Free System Cells Cellular Structures Cessation of life Characteristics Client Collaborations Complex Cryoelectron Microscopy Crystallography Cytoplasm DNA Diabetes Mellitus Dissociation Estrogen Receptors Eukaryotic Cell Family Feedback Fluorescence Genes Genetic Transcription I Kappa B-Alpha Immune response Inflammatory Kinetics Label Ligand Binding Domain Literature Macromolecular Complexes Malignant Neoplasms Mammalian Cell Membrane Methods Molecular Molecular Chaperones Movement Nuclear Phosphorylation Phosphotransferases Preparation Process Proteins Protocols documentation RNA Reporting Research Rest Roentgen Rays Signal Transduction Structure System Techniques Ubiquitination Untranslated RNA Viral Virus Diseases biological adaptation to stress biophysical techniques cell growth inhibitor innovation macromolecule member overexpression physical state reconstitution response solid state transcription factor

项目摘要

The major overall aim of the research described in this MIRA proposal is to elucidate the structural and dynamic characteristics of large macromolecules and their complexes, which generally contain both well- structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems have been under study in our lab for several years. Members of the NFκB family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (IκBs) until the cell receives an external signal. NFκB is activated by phosphorylation and ubiquitination of the IκBα, which is then targeted for proteasomal degradation, releasing the NFκB to translocate into the nucleus. In a classical negative feedback mechanism, NFκB upregulates transcription of IκBα in addition to signal-specific stress-response genes: newly-synthesized IκBα kinetically enhances NFκB dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process and in an exciting new observation, it was recently shown that the stability of the resting NFκB-IκBα complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NFκB-IκBα-DNA complex formed in the nucleus during molecular stripping. We propose the structural characterization of this NFκB-IκBα-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between the binary and ternary complexes of NFκB, IκBα and DNA, and the ternary NFκB-IκBα-RNA complex using specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts containing the over-expressed, folded co-chaperones required according to literature reports for the formation of a stable complex with Hsp90. The advantage of this cell-free system is that it can be tuned to the optimization of complex formation by varying the relative amounts of the component cell extracts.

在这个MIRA提案中描述的研究的主要总体目标是阐明结构和

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Characterization of the High-Affinity Fuzzy Complex between the Disordered Domain of the E7 Oncoprotein from High-Risk HPV and the TAZ2 Domain of CBP.

DOI：
10.1021/acs.biochem.1c00669
发表时间：
2021-12-28
期刊：
Biochemistry
影响因子：
2.9
作者：
Risør MW;Jansma AL;Medici N;Thomas B;Dyson HJ;Wright PE
通讯作者：
Wright PE

Early Strides in NMR Dynamics Measurements.

DOI：
10.1021/acs.biochem.1c00141
发表时间：
2021-11-23
期刊：
Biochemistry
影响因子：
2.9
作者：
Dyson HJ
通讯作者：
Dyson HJ

Thermodynamic Stability and Aggregation Kinetics of EF Helix and EF Loop Variants of Transthyretin.

DOI：
10.1021/acs.biochem.1c00073
发表时间：
2021-03-16
期刊：
Biochemistry
影响因子：
2.9
作者：
Ferguson JA;Sun X;Dyson HJ;Wright PE
通讯作者：
Wright PE

Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.

DOI：
10.1021/acs.biochem.2c00528
发表时间：
2022-12-06
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Krois, Alexander S.;Park, Sangho;Martinez-Yamout, Maria A.;Dyson, H. Jane;Wright, Peter E.
通讯作者：
Wright, Peter E.

Backbone and side-chain chemical shift assignments of p50 subunit of NF-κB transcription factor.