Mapping and controlling the segmental dynamic networks of Cyclophilin A as they r

绘制和控制亲环蛋白 A 的分段动态网络

• 批准号: 8781571
• 负责人: Michael Joseph Holliday
• 金额: $ 2.92万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781571
• 关键词: Active Sites; Allosteric Site; Automobile Driving; Binding; Binding Sites; Biological Process; Biomolecular Nuclear Magnetic Resonance; Breast; Catalysis; Chemoprotection; Chimeric Proteins; Chronic Myeloid Leukemia; Clinical; Collection; Colorectal; Communication; Complex; Computing Methodologies; Coupled; Coupling; Cyclophilin A; Cyclophilins; Cyclosporine; Cytokine Signaling; Data; Data Analyses; Development; Disease; Disease Progression; Distal; Distant; Drug Design; Enzymes; Exhibits; Face; Figs - dietary; Future; HIV; HIV-1; HIV-1 protease; Human; Immune response; In Vitro; Inflammatory; Kinetics; Link; Liver; Lung; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Methodology; Methods; Modeling; Molecular Chaperones; Molecular Conformation; Motion; Movement; Mutation; NMR Spectroscopy; Neoplasm Metastasis; Nuclear Magnetic Resonance; Pancreas; Pathway interactions; Peptidylprolyl Isomerase; Prostate; Protein Dynamics; Protein Region; Proteins; RNA-Directed DNA Polymerase; Relaxation; Research; Resolution; Role; Sampling; Signal Transduction; Site; Solutions; Structure; System; T-Cell Proliferation; Techniques; Therapeutic; Thermodynamics; Work; analytical method; base; bcr-abl Fusion Proteins; biophysical techniques; cancer therapy; design; improved; inhibitor/antagonist; melanoma; millisecond; molecular dynamics; mutant; novel; novel strategies; prevent; protein function; public health relevance; research study; therapeutic development; therapeutic protein; therapeutic target

DESCRIPTION (provided by applicant): Current rational, structure based approaches to designing therapeutic protein inhibitors are, largely, limited to targeting the active site of a sigle low energy conformation of a protein. As proteins in solution are continually sampling a structurally heterogeneous range of conformations and allosteric effects have often been shown to influence function distant from the active site, significant improvement to rational drug design could be realized by advancing our understanding of what dynamic motions exist in proteins and how they relate to function. In humans, Cyclophilin A (CypA), a peptidyl-prolyl isomerase, is involved in a range of biological function, including chaperone activity, intercellular signaling, and cytokine signaling, as well as having roles, both cytosolic and extracellularly, in driving development, progression, metastasis, and chemoprotection in a number of cancers, including lung, breast, colorectal, prostate, and liver. This study aims to measure dynamics in CypA and to unravel how these motions communicate throughout the protein to control enzymatic function. More broadly, the proposed work will develop several novel methodologies for studying this crucial dynamics-function relationship, which will be applicable to many protein systems. For the proposed studies, nuclear magnetic resonance (NMR) spectroscopy relaxation experiments will provide atomic resolution measurement of dynamic motions across timescales from picoseconds to milliseconds, including, critically, quantitative measurement of exchange rates near the timescale of catalysis (?s-ms). Additionally, several biophysical techniques, including NMR, ITC, and CD will be used to functionally characterize proteins in vitro. Two novel approaches will then be carried out to probe the dynamics-function relationship in CypA. In the first, residues, distal from the protein active site, will be identified for which dynamics are eiter altered by other CypA mutants or altered during catalysis (with of saturating concentrations of a model substrate), indicating that they are still within the functionally relevant networks of dynamic communication. Single site mutations of these residues will permit alteration of the global dynamics of the protein independent of structural changes to the active site and analysis of the functional implications of given dynamic changes. Multiple 'dynamic mutants', when analyzed in parallel, will reveal the networks through which regions of the protein communicate, as well as the motions involved in regulating function. The second approach will utilize recent advances in NMR data-driven molecular dynamics (MD) simulations to generate conformationally broad ensembles of CypA alone, during catalysis, and with dynamics altering mutations. A novel analytical technique, which allows for identification of partially correlated localization within these MD ensembles, will allow us to map, at high resolution, the specific pathways of communication in CypA. Combined, these complementary approaches will bring us closer to understanding how variable dynamics are communicated and how they relate to function, a critical step in advancing structure/dynamics based drug design.

DESCRIPTION (provided by applicant): Current rational, structure based approaches to designing therapeutic protein inhibitors are, largely, limited to targeting the active site of a sigle low energy conformation of a protein. As proteins in solution are continually sampling a structurally heterogeneous range of conformations and allosteric effects have often been shown to influence function distant from the active site, significant improvement to rational drug design could be realized by advancing our understanding of what dynamic motions exist in proteins and how they relate to function. In humans, Cyclophilin A (CypA), a peptidyl-prolyl isomerase, is involved in a range of biological function, including chaperone activity, intercellular signaling, and cytokine signaling, as well as having roles, both cytosolic and extracellularly, in driving development, progression, metastasis, and chemoprotection in a number of cancers, including lung, breast, colorectal, prostate, and liver. This study aims to measure dynamics in CypA and to unravel how these motions communicate throughout the protein to control enzymatic function. More broadly, the proposed work will develop several novel methodologies for studying this crucial dynamics-function relationship, which will be applicable to many protein systems. For the proposed studies, nuclear magnetic resonance (NMR) spectroscopy relaxation experiments will provide atomic resolution measurement of dynamic motions across timescales from picoseconds to milliseconds, including, critically, quantitative measurement of exchange rates near the timescale of catalysis (?s-ms). Additionally, several biophysical techniques, including NMR, ITC, and CD will be used to functionally characterize proteins in vitro. Two novel approaches will then be carried out to probe the dynamics-function relationship in CypA. In the first, residues, distal from the protein active site, will be identified for which dynamics are eiter altered by other CypA mutants or altered during catalysis (with of saturating concentrations of a model substrate), indicating that they are still within the functionally relevant networks of dynamic communication. Single site mutations of these residues will permit alteration of the global dynamics of the protein independent of structural changes to the active site and analysis of the functional implications of given dynamic changes. Multiple 'dynamic mutants', when analyzed in parallel, will reveal the networks through which regions of the protein communicate, as well as the motions involved in regulating function. The second approach will utilize recent advances in NMR data-driven molecular dynamics (MD) simulations to generate conformationally broad ensembles of CypA alone, during catalysis, and with dynamics altering mutations. A novel analytical technique, which allows for identification of partially correlated localization within these MD ensembles, will allow us to map, at high resolution, the specific pathways of communication in CypA. Combined, these complementary approaches will bring us closer to understanding how variable dynamics are communicated and how they relate to function, a critical step in advancing structure/dynamics based drug design.