Introducing a fluorescence-based standard to quantify protein partitioning into membranes

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Peripheral membrane protein affinity for lipid bilayers is crucial for cellular functions.
• Existing methods for determining the partition coefficient (KP) have limitations.
• A robust and versatile method is needed for accurate KP measurement.

Purpose of the Study:

• To develop a novel, versatile method for determining the partition coefficient (KP) of peripheral membrane proteins.
• To establish a simple measurement standard using eGFP-His6 and NTA(Ni)-functionalized GUVs.
• To compare two measurement modes (single molecule and imaging) for KP determination.

Main Methods:

• Developed a method combining single molecule detection and fluorescence imaging.
• Utilized fluorescence correlation spectroscopy (FCS) in single molecule mode.
• Employed confocal fluorescence imaging and semi-automated software analysis in imaging mode.
• Used giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids and eGFP-His6 as a standard.

Main Results:

• Achieved equivalent KP values between single molecule and imaging modes.
• Demonstrated a stronger-than-linear increase in eGFP-His6 affinity with increasing NTA(Ni) lipid concentration.
• The novel method accurately determined the KP of the MARCKS effector domain, consistent with prior studies.

Conclusions:

• The developed dual-mode method provides a versatile and accurate approach for measuring peripheral membrane protein affinity (KP).
• This method overcomes limitations of previous techniques and captures complex binding trends.
• Offers a practical manual for KP measurement and interpretation.