Examination of conformational dynamics of AdiC transporter with fluorescence-polarization microscopy.

IF 3.3 2区 医学 Q1 PHYSIOLOGY
Journal of General Physiology Pub Date : 2025-05-05 Epub Date: 2025-02-20 DOI:10.1085/jgp.202413709
John H Lewis, Yufeng Zhou, Zhe Lu
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引用次数: 0

Abstract

To understand the mechanism underlying the ability of individual AdiC molecules to transport arginine and agmatine, we used a recently developed high-resolution single-molecule fluorescence-polarization microscopy method to investigate conformation-specific changes in the emission polarization of a bifunctional fluorophore attached to an AdiC molecule. With this capability, we resolved AdiC's four conformations characterized by distinct spatial orientations in the absence or presence of the two substrates, and furthermore, each conformation's two energetic states, totaling 24 states. From the lifetimes of individual states and state-to-state transition probabilities, we determined 60 rate constants characterizing the transitions and 4 KD values characterizing the interactions of AdiC's two sides with arginine and agmatine, quantitatively defining a 24-state model. This model satisfactorily predicts the observed Michaelis-Menten behaviors of AdiC. With the acquired temporal information and existing structural information, we illustrated how to build an experiment-based integrative 4D model to capture and exhibit the complex spatiotemporal mechanisms underlying facilitated transport of substrates. However, inconsistent with what is expected from the prevailing hypothesis that AdiC is a 1:1 exchanger, all observed conformations transitioned among themselves with or without the presence of substrates. To corroborate this unexpected finding, we performed radioactive flux assays and found that the results are also incompatible with the hypothesis. As a technical advance, we showed that a monofunctional and the standard bifunctional fluorophore labels report comparable spatial orientation information defined in a local frame of reference. Here, the successful determination of the complex conformation-kinetic mechanism of AdiC demonstrates the unprecedented resolving power of the present microscopy method.

用荧光偏振显微镜检查AdiC转运体的构象动力学。
为了了解单个AdiC分子运输精氨酸和胍丁氨酸能力的机制,我们使用了最近开发的高分辨率单分子荧光偏振显微镜方法来研究附着在AdiC分子上的双功能荧光团的发射偏振的构象特异性变化。利用这种能力,我们分析了在没有或存在两种底物的情况下,AdiC的四个具有不同空间取向的构象,并进一步分析了每个构象的两个能态,共计24个态。从单个状态的寿命和状态到状态的转变概率,我们确定了表征转变的60个速率常数和表征AdiC两侧与精氨酸和agmatine相互作用的4个KD值,定量定义了一个24态模型。该模型令人满意地预测了观测到的AdiC的Michaelis-Menten行为。利用获取的时间信息和现有的结构信息,我们说明了如何构建基于实验的综合四维模型来捕捉和展示基质促进运输的复杂时空机制。然而,与普遍假设的预期不符,即AdiC是1:1的交换剂,所有观察到的构象在有或没有底物存在的情况下都在它们之间转换。为了证实这一意想不到的发现,我们进行了放射性通量分析,发现结果也与假设不相容。作为一项技术进步,我们展示了单功能和标准双功能荧光团标签报告在局部参考框架中定义的可比空间方向信息。在这里,成功地确定了AdiC的复杂构象-动力学机制,证明了当前显微镜方法的前所未有的分辨能力。
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来源期刊
CiteScore
6.00
自引率
10.50%
发文量
88
审稿时长
6-12 weeks
期刊介绍: General physiology is the study of biological mechanisms through analytical investigations, which decipher the molecular and cellular mechanisms underlying biological function at all levels of organization. The mission of Journal of General Physiology (JGP) is to publish mechanistic and quantitative molecular and cellular physiology of the highest quality, to provide a best-in-class author experience, and to nurture future generations of independent researchers. The major emphasis is on physiological problems at the cellular and molecular level.
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