Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment

Abstract

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Figure 1

(a) Schematic representation of the preparation of a supported cell membrane electrophoresis platform. (b) (Left) Fluorescence images of the GLUT1 complex (0.1× PBS) within membrane patches at various pH values under membrane electrophoresis, with a 10 μm scale bar. (Right) The corresponding normalized intensity profiles and displacement of fluorescence intensity center at different pH values. (c) (Left) Fluorescence images of Fast-DiO (0.1× PBS) at different pH values under membrane electrophoresis, with a 10 μm scale bar. (Right) The corresponding normalized intensity profiles and displacement of fluorescence intensity center at different pH values.

Figure 2

Proposed force model of a transmembrane protein labeled with antibodies in the supported cell membrane.

Figure 3

(a) Electrophoretic mobilities of the GLUT1 complex at pH 4, pH 7.4, and pH 10 under various PBS buffer concentrations (n = 30 from three independent experiments; 10 patches per experiment). (b) The results of nonlinear curve fitting at pH 4, 7.4, and pH 10. (c) Calculated forces that acted on the GLUT1 complex at pH 4, 7.4, and pH 10. Electroosmotic force (F_EO): blue circle. Total drag force (F_{drag_M} + F_{drag_W}): orange circle. Electric force on portion B (F_EB): yellow circle. Electric force on portion A (F_EA): pink circle. Total driving force (F_EO + F_EA + F_EB): purple circle.

Figure 4

(a) Electrophoretic mobilities of Fast-DiO at pH 4, pH 7.4, and pH 10 under different PBS buffer concentrations (n = 60 from six independent experiments; 10 patches per experiment). (b) The result of nonlinear curve fitting at pH 4, 7.4, and pH 10. (c) Calculated forces that acted on the Fast-DiO at pH 4, 7.4, and pH 10. Electroosmotic force (F_EO): blue circle. Total drag force (F_{drag_M} + F_{drag_W}): orange circle. Electric force on portion B (F_EB): yellow circle. Electric force on portion A (F_EA): pink circle. Total driving force (F_EO + F_EA + F_EB): purple circle.

Figure 5

(Left) Fluorescence images depicting the GLUT1 complex and Fast-DiO within cell membrane patches, showcasing their behaviors in two distinct buffer environments during membrane electrophoresis. Scale bar: 10 μm. (Right) Normalized intensity profiles of the GLUT1 complex and Fast-DiO captured at 0 and 180 s after the initiation of membrane electrophoresis.

Figure 6

Schematic illustration of how rapidly moving charged membrane species affect the migration of the GLUT1 complex at different pH levels: (a) pH 4, (b) pH 7.4, and (c) pH 10. The yellow arrow is the intrinsic electric force exerted on portion B due to the intrinsic charge of portion B. The gray arrow is the induced electric force caused by the polarization of the rapidly moving membrane species. The effective electric force applied on portion B is the summation of the intrinsic electric force and the induced electric force. The blue arrow indicates the electroosmotic flow.

Figure 7

Predicted electrophoretic mobilities of transmembrane proteins at varying (a) r_B (constant r_A, q_A, q_B), (b) r_B (constant r_A, q_A, V_B/q_B), (c) r_A (constant r_B, q_A, q_B), (d) r_A (constant r_B, q_B, V_A/q_A). The dashed line indicates the Debye length at 1×, 0.1× 0.01×, 0.001×, and 0.0001× PBS (from left to right).