In-Cell Fast Photochemical Oxidation Interrogates the Native Structure of Integral Membrane Proteins.

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Integral membrane proteins (IMPs) are crucial for cellular processes but difficult to study.
• Existing methods often require cell lysis or lack in situ capabilities.

Purpose of the Study:

• To introduce a novel method, in-cell fast photochemical oxidation of membrane proteins (IC-FPOMP), for in situ footprinting of IMPs in live cells.
• To demonstrate the utility of IC-FPOMP for investigating IMP structure-function relationships and facilitating drug discovery.

Main Methods:

• IC-FPOMP utilizes reactive oxygen radicals generated near the cell membrane.
• A laser and a 96-well plate platform enable high-throughput and rapid footprinting.
• The method was applied to human glucose transporter-hGLUT1 and human gamma-glutamyl carboxylase-hGGCX.

Main Results:

• IC-FPOMP successfully mapped both transmembrane and extramembrane regions of IMPs in live cells.
• Conformational differences of hGLUT1 in liposomes versus cells were observed.
• In-cell drug screening revealed in vivo drug-binding behavior for hGLUT1.

Conclusions:

• IC-FPOMP provides unprecedented insights into IMP structure-function relationships within their native cellular environment.
• This method accelerates IMP research and opens new avenues for in-cell drug screening and discovery.