High Resolution Single Molecule Analysis of Fast Folding and its Coupling to Binding

快速折叠及其耦合耦合的高分辨率单分子分析

基本信息

  • 批准号:
    1616759
  • 负责人:
  • 金额:
    $ 70.39万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Continuing Grant
  • 财政年份:
    2016
  • 资助国家:
    美国
  • 起止时间:
    2016-08-15 至 2019-07-31
  • 项目状态:
    已结题

项目摘要

Proteins are the cellular nanomachines in charge of most biological functions, including energy production, DNA replication and transcription, enzymatic catalysis, signaling, cellular scaffolding and defense. But proteins are also inherently flexible polymers that must fold into the complex native 3D structures corresponding to their biologically functional states in a self-assembly process determined by the chemical blueprints encoded in their amino acid sequence. Thus the mechanisms by which proteins fold and function are a critical component of almost every aspect of molecular and cell biology. Understanding the intertwined mechanisms of folding and function also brings about the opportunity to predict, engineer, and design biological function "a la carte", thus conveying unparalleled transformative impact to Society. Moreover, because proteins are at the lowest echelon of biological complexity where Biology effectively meets Physics, Chemistry, and Engineering, their study constitutes an ideal arena for training the new generations of multidisciplinary researchers, preparing them for the emerging fields of Quantitative and Synthetic Biology. Activities are designed around a team-based structure aimed at facilitating integration of members at various levels of education ranging from postdoctoral fellows and graduate students to undergraduate and high school students/teachers. An equally important element of this project is the strong commitment to participation of underrepresented groups in research. The PI will actively recruit project members from several existing research mentoring programs for underrepresented minorities in STEM fields to participate in the research activities of this project.A major drive for modern protein research has been to develop experimental methods to resolve the myriads of pathways and complex mechanisms that are predicted by advanced theory and atomistic molecular simulations. Detecting such inherent kinetic complexity in experiments has remained elusive, even with modern methods that exhibit improved time, structural, or single-molecule resolution. The overall objective of this project is to bridge this gap by experimentally monitoring the transition paths of individual protein molecules as they fold. Advanced single-molecule fluorescence methods will be used in conjunction with theoretical and computational analyses to measure transition paths and folding mechanisms of fast-folding protein domains. Fast-folding domains are optimal targets because their marginal cooperativity ensures significant populations of "excited" states and, somewhat counterintuitively, slower transition paths over their broad, shallow folding barriers. Moreover, the microsecond folding of these domains facilitates direct comparison with modern atomistic simulations. To reach the required resolution, we rely on approaches we recently developed for achieving microsecond resolution single-molecule fluorescence detection, such as better photoprotection systems and procedures for maximum likelihood analysis of photon arrival times, together with implementation of 2-color and 3-color FRET schemes to measure multiple distances. Through the realization of these experiments and computational analyses on select fast-folding domains we will investigate the structural, sequence, and environmental determinants of the mechanisms for folding. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.
蛋白质是负责大多数生物功能的细胞纳米机器,包括能量产生、DNA 复制和转录、酶催化、信号传导、细胞支架和防御。 但蛋白质本质上也是柔性聚合物,必须在自组装过程中折叠成与其生物功能状态相对应的复杂天然 3D 结构,该自组装过程由其氨基酸序列中编码的化学蓝图决定。因此,蛋白质折叠和发挥作用的机制是分子和细胞生物学几乎每个方面的关键组成部分。了解折叠和功能相互交织的机制也带来了“按菜单点菜”预测、工程和设计生物功能的机会,从而为社会带来无与伦比的变革性影响。此外,由于蛋白质处于生物复杂性的最低梯队,生物学有效地满足了物理、化学和工程学,因此他们的研究构成了培训新一代多学科研究人员的理想舞台,为他们进入定量和合成生物学的新兴领域做好准备。 活动是围绕基于团队的结构设计的,旨在促进从博士后研究员和研究生到本科生和高中生/教师等不同教育层次的成员的融合。该项目的一个同样重要的要素是对代表性不足的群体参与研究的坚定承诺。 PI将积极从现有的几个针对STEM领域代表性不足的少数群体的研究指导计划中招募项目成员来参与该项目的研究活动。现代蛋白质研究的一个主要驱动力是开发实验方法来解决由先进理论和原子分子模拟预测的无数途径和复杂机制。即使使用表现出改进的时间、结构或单分子分辨率的现代方法,在实验中检测这种固有的动力学复杂性仍然难以捉摸。该项目的总体目标是通过实验监测单个蛋白质分子折叠时的转变路径来弥补这一差距。先进的单分子荧光方法将与理论和计算分析结合使用,以测量快速折叠蛋白质结构域的转变路径和折叠机制。快速折叠域是最佳目标,因为它们的边际协同性确保了大量的“兴奋”状态,并且在某种程度上违反直觉的是,它们在宽而浅的折叠势垒上的过渡路径较慢。此外,这些域的微秒折叠有助于与现代原子模拟进行直接比较。为了达到所需的分辨率,我们依靠最近开发的实现微秒分辨率单分子荧光检测的方法,例如更好的光保护系统和光子到达时间最大似然分析程序,以及实施 2 色和 3 色 FRET 方案来测量多个距离。通过对选定的快速折叠域进行这些实验和计算分析,我们将研究折叠机制的结构、序列和环境决定因素。该项目由分子和细胞生物科学部的分子生物物理学集群和物理部的生命系统物理学项目联合资助。

项目成果

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Victor Munoz其他文献

Engrailed homeodomain uses an electrostatic spring-loaded mechanism to change conformation upon binding to DNA
  • DOI:
    10.1016/j.bpj.2021.11.2619
  • 发表时间:
    2022-02-11
  • 期刊:
  • 影响因子:
  • 作者:
    Benjamin Tanielian;Nicola D'Amelio;Mourad Sadqi;Victor Munoz
  • 通讯作者:
    Victor Munoz
Catching Fast Protein Folding in the Act: Resolving (Un)Folding Transition Paths using Advanced Single-molecule Spectroscopy
  • DOI:
    10.1016/j.bpj.2019.11.1041
  • 发表时间:
    2020-02-07
  • 期刊:
  • 影响因子:
  • 作者:
    Nivin Mothi;Mourad Sadqi;Victor Munoz
  • 通讯作者:
    Victor Munoz
The structure of cosmic strings of a U(1) gauge field for the conservation of B - L
用于 B - L 守恒的 U(1) 规范场的宇宙弦结构
Transcriptional factors control their diffusion on DNA by modulating their dynamics
  • DOI:
    10.1016/j.bpj.2021.11.492
  • 发表时间:
    2022-02-11
  • 期刊:
  • 影响因子:
  • 作者:
    Rama Reddy Goluguri;Mourad Sadqi;Victor Munoz
  • 通讯作者:
    Victor Munoz
Biophysical studies of the promiscuous transcription factor engrailed: Proposed mechanism for the conformational response by electrostatics and DNA recognition
  • DOI:
    10.1016/j.bpj.2023.11.1264
  • 发表时间:
    2024-02-08
  • 期刊:
  • 影响因子:
  • 作者:
    Benjamin Tanielian;Mourad Sadqi;Victor Munoz
  • 通讯作者:
    Victor Munoz

Victor Munoz的其他文献

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{{ truncateString('Victor Munoz', 18)}}的其他基金

Dissecting the Rate Theory for Protein Folding Dynamics via Advanced Single-Molecule Fluorescence Experiments
通过先进的单分子荧光实验剖析蛋白质折叠动力学的速率理论
  • 批准号:
    2112710
  • 财政年份:
    2021
  • 资助金额:
    $ 70.39万
  • 项目类别:
    Continuing Grant
CREST Center for Cellular and Biomolecular Machines
CREST 细胞和生物分子机器中心
  • 批准号:
    2112675
  • 财政年份:
    2021
  • 资助金额:
    $ 70.39万
  • 项目类别:
    Continuing Grant
CREST Center for Cellular and Biomolecular Machines
CREST 细胞和生物分子机器中心
  • 批准号:
    1547848
  • 财政年份:
    2016
  • 资助金额:
    $ 70.39万
  • 项目类别:
    Continuing Grant
Experimental Investigations of Protein Reconfiguration Dynamics
蛋白质重构动力学的实验研究
  • 批准号:
    0317294
  • 财政年份:
    2003
  • 资助金额:
    $ 70.39万
  • 项目类别:
    Continuing Grant

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Acquisition of Two-Color 3-D Minflux for Live-Cell Single Molecule Imaging and Tracking at Unprecedented Spatial and Temporal Resolution
采集双色 3-D Minflux,以前所未有的空间和时间分辨率进行活细胞单分子成像和跟踪
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