Quantitative Membrane Paramagnetic Relaxation Enhancement (mPRE) Measurements on Lipid-Anchored Proteins: Insight...

Area of Science:

• Biophysics
• Structural Biology
• Membrane Protein Dynamics

Background:

• Lipid-anchored eukaryotic proteins require specific membrane orientations for function.
• Accurate mapping of protein orientations is crucial but technically challenging.
• Existing membrane paramagnetic relaxation enhancement (mPRE) NMR methods are distorted for irreversibly lipid-anchored proteins.

Purpose of the Study:

• To develop a novel strategy for quantifying membrane orientations of lipidated proteins.
• To overcome limitations of mPRE NMR for irreversibly anchored proteins.
• To investigate the orientational dynamics of KRas4B and HRas oncoprotein membranes.

Main Methods:

• Developed a tunable lipidation strategy using synthetic alk-1-en-3-one reagents.
• Conjugated undec-1-en-3-one to proteins to create a fast-exchange equilibrium for mPRE.
• Applied artifact-free mPRE NMR to a ubiquitin model system, KRas4B, and HRas.

Main Results:

• The new method restores the theoretical dependence between apparent Γ2 rates and interspin distances in mPRE.
• Negatively charged POPS indirectly influences KRas4B orientations by constraining its C-terminal region.
• HRas on neutral membranes adopts an orientation similar to KRas4B on anionic membranes, suggesting orientational convergence.

Conclusions:

• Established a generalizable method for quantifying membrane orientations of lipidated proteins.
• Provided new insights into Ras protein regulation and membrane interactions.
• Enabled the study of previously intractable lipid-anchored protein systems.