Reconstitution of Functionalized Transmembrane Domains of Receptor Proteins into Biomimetic Membranes
Daniel R Scott, Vitalii Silin, Hirsh Nanda
For integral membrane proteins, an assessment of their structures and interactions within a biomimetic lipid bilayer environment is critical for evaluating their cellular function. Hydrophobic sequences prevalent within the transmembrane domain(s), however, make these proteins susceptible to aggregation, and thus create difficulties in examining their structural and functional properties via canonical techniques. Working exclusively with single-pass transmembrane (TM) segments of bitopic membrane proteins ¿ in the form of soluble peptides ¿ bypasses many of the pitfalls of full-length protein preparations, while allowing for the opportunity to examine the properties of TM domains within bio-mimetic membrane environments. In this study, peptides mimicking the TM domains of the epidermal growth factor (EGFR) and CD4 receptors, both cell-signaling surface receptors, have been reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers. The formation of their native α-helical structures within vesicle membranes was observed from circular dichroism, and full partition of the peptides into the membrane was demonstrated by tryptophan fluorescence and neutron reflectivity (NR). Utilizing an engineered planar lipid bilayer system ideal for surface characterization methods such as surface plasmon resonance and NR, the TM peptides ¿ functionalized with a N-terminal biotin tag ¿ proved capable of ¿activating¿ a membrane surface as evidenced by the capture of streptavidin. Prospectively, these TM peptides may be ligated with their extra-membrane domains by means of bioengineering methods. These reconstituted near-native membrane protein systems could be utilized to advance our biophysical understanding of the mechanistic processes of cell signal transduction, and in formulating surfaces for powerful biosensor applications.
membrane protein structure, protein-membrane interactions, tryptophan fluorescence, SPR, neutron reflectivity
, Silin, V.
and , H.
Reconstitution of Functionalized Transmembrane Domains of Receptor Proteins into Biomimetic Membranes, Langmuir, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=918575
(Accessed December 1, 2023)