相关实验视频

Updated: May 28, 2025

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Published on: March 14, 2019

一种基于光的温度跳跃装置,用于在毫秒时间尺度上说明蛋白质动力学

Liang-Che Kung¹, Li-Kang Chu¹

¹Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Road, Hsinchu 300044, Taiwan.

Analytical chemistry

|February 13, 2025

概括

一种新的基于光的温度跳跃 (T-跳跃) 方法揭示了毫秒时间尺度上的蛋白质动态. 该技术使用最小的样本体积量来量化牛血清白蛋白 (BSA) 等蛋白质中的大域运动.

科学领域:

生物物理学的生物物理.
蛋白质动力学蛋白质动力学
频谱学是一种光谱学.

背景情况:

了解蛋白质动态对于阐明生物功能至关重要.
现有的技术往往缺乏在生物学上相关的时间尺度上捕捉大域运动的分辨率.
毫秒时间尺度的动态弥合了快速局部运动和稳定状态形态之间的差距.

研究的目的:

开发和验证基于光的温度跳跃 (T-jump) 模块,用于研究蛋白质动力学.
在毫秒时间尺度上研究蛋白质的大域运动.
在生理温度范围内对热刺激的蛋白质反应的特征.

主要方法:

一个基于光的T跳模块,使用1467nm二极管激光和"光学里曼和值"进行快速加热 (5.0°C在~2 ms).
通过时间解析的光强度变化监测溶解的托芬的温度演变.
采用微点镜头用于小样本体积 (1μL) 和自制的恒温用于温度控制.

主要成果:

成功证明了在生理温度 (35.0-39.9°C) 内的毫秒分辨率T跳测量.
描述了牛血清白蛋白 (BSA) 的动态,其明显激活能量为276 ± 23 kJ mol-1.1.
人类血清白蛋白 (HSA) 在测试条件下没有表现出可辨认的动态成分.

结论:

开发的毫秒分辨率T跳技术有效地说明了大域蛋白质动态.
这种方法弥合了快速 (ns-μs) 和稳定状态蛋白质动态特性之间的差距.
该技术对于研究生物物理研究中的蛋白质构成变化和热反应具有价值.

更多相关视频

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Published on: October 9, 2014

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Published on: May 30, 2021

相关概念视频

Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

Protein Diffusion in the Membrane

Protein Diffusion in the Membrane

Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...