Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles

Area of Science:

• Biophysics
• Structural Biology
• Protein-Membrane Interactions

Background:

• Amphitropic protein binding to membranes is key to cellular processes and disease.
• Protein misfolding and aggregation are linked to membrane interactions.
• A-Cage-C, derived from α-Lactalbumin, is known to cause membrane leakage.

Purpose of the Study:

• Elucidate conformational changes in A-Cage-C that promote bilayer interaction.
• Understand the structural basis of protein-membrane binding and potential aggregation.

Main Methods:

• Expression and purification of double-labelled A-Cage-C.
• Use of partially deuterated bicelles as a membrane model.
• Investigation at non-binding (pH 7.0) and binding (pH 4.5) conditions.
• In silico analyses, Nuclear Magnetic Resonance (NMR), conformational clustering, and Molecular Dynamics (MD).

Main Results:

• A-Cage-C exhibits dynamic conformations in both bound and unbound states.
• Identified a specific conformational cluster likely representing the binding state.
• Observed unfolding around the W23 segment and reorientation at pH 4.5.
• Evidence for overall protein elongation upon interaction with model bilayers.

Conclusions:

• Unfolding and reorientation of the W23 segment are likely necessary for A-Cage-C intercalation into bilayers at pH 4.5.
• Protein elongation is observed in the presence of model membranes.
• Conformational flexibility is central to A-Cage-C's membrane interaction mechanism.