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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...

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Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

Published on: January 6, 2023

Microscale fluorescent thermal stability assay for membrane proteins.

Alexander I Alexandrov¹, Mauro Mileni, Ellen Y T Chien

¹Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Structure (London, England : 1993)

|March 13, 2008

Summary

This study presents a new microscale fluorescent stability screen for membrane proteins. The method uses N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) to assess protein integrity and guide biophysical characterization.

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Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Published on: April 28, 2011

Area of Science:

Biochemistry
Structural Biology
Membrane Protein Research

Background:

Membrane protein stability studies are challenging due to detergent interference in biophysical characterization.
Technical difficulties limit systematic analysis of membrane protein behavior under various solution conditions.

Purpose of the Study:

To introduce an efficient microscale fluorescent stability screen for membrane proteins.
To enable detailed stability profiling under diverse solution and ligand conditions.
To guide subsequent purification, biophysical characterization, and crystallization efforts.

Main Methods:

Utilized the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM).
Employed the chemical reactivity of native internal cysteines as a sensor for protein structural integrity.
Applied the screen to analyze three protein families, including the Apelin G protein-coupled receptor (APJ).

Main Results:

Demonstrated an efficient microscale fluorescent stability screen for membrane proteins.
Successfully profiled membrane protein stability under various conditions.
Generated thermal stability data to inform protein handling and characterization.

Conclusions:

The CPM-based fluorescent screen is effective for membrane protein stability assessment.
This method overcomes detergent-related challenges in biophysical characterization.
The stability information aids in optimizing purification, characterization, and crystallization of membrane proteins.