Abstract
Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower Kd ) as the proportion of anionic lipid increases. Interestingly, at high proportions of phosphatidylserine, the Kd values of all four proteins began to converge. We also found that although koff values for all four proteins followed trends similar to those observed for the Kd values, the variation among the proteins was much lower, indicating that much of the variation came from the kinetic binding (kon) of the proteins. These findings indicate that the combination of silicon photonic microring resonator arrays and nanodiscs enables rapid interrogation of biomolecular binding interactions at model cell membrane interfaces.
Highlights
Plasmon resonance, can be a time-consuming and complicated process that often requires large quantities of expensive reagents. This holds especially true for monitoring interactions at the cell membrane surface, which plays a critical role in the regulation of important biological processes such as cell signaling and blood coagulation, the latter of which involves protein– protein and protein–lipid interactions, and is of particular relevance to this study
We combined the versatility of nanodiscs with highly multiplexible silicon photonic microring resonators to study protein–lipid interactions involved in the blood coagulation cascade
PT, factor X (fX), fVIIa, and activated protein C (APC) bind to the membrane surface through their GLA domains [24, 25]
Summary
In previous work [23], a four-channel microfluidic chamber was used to direct solutions of four nanodiscs, each having a different lipid composition, across different regions of a microring resonator substrate to create a four-component sensing array. Because the nanodiscs naturally physisorb to the silicon oxide surface, neither the surface nor the nanodiscs were modified for attachment Arrays of these nanodiscs with seven different lipid compositions were used to determine both the equilibrium dissociation constant, Kd, and kinetic dissociation rate, koff, for interactions with four different proteins involved in the blood coagulation pathway: PT, fX, fVIIa, and APC. Because all of the coagulation proteins studied bind in a Ca2ϩ-dependent manner, the nanodisc array could be completely regenerated by flowing a Ca2ϩ-free buffer, followed by interrogation of subsequent coagulation protein–lipid interactions In this way, identical titrations for fX, fVIIa, and APC were performed and can be found in supplemental Figs. Both fVIIa and APC had the lower koff values when comparing PA to PS containing nanodiscs with koff values of 50% PA nanodiscs of 0.344 and 0.293 minϪ1, compared with 0.51 and 0.67 minϪ1 for 50% PS, respectively (supplemental Table S3)
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