Tunable nucleation time of functional sphingomyelinase--lipid features studied by membrane array statistic tool.

Aggregation or assembly of lipids and proteins could significantly change the proteins' function. A peripheral membrane enzyme, sphingomyelinase (SMase), has been reported to be able to assemble to a functional feature with its lipid substrate, sphingomyelin (SM), and its lipid product, ceramide (Cer). SMase seems to processes its substrate more effectively in this feature. Here, we report that the functional feature has a tunable formation time. The peculiar behavior is that the feature formation has a time lag depending on the membrane composition. We hypothesized that the time lag is due to the significant nucleation energy barrier when the feature phase forms in its metastable parent phase in the 2-D lipid membrane. To study the stochastic nucleation of the feature, we built a corralled lipid membrane platform with numerous isolated membrane systems in parallel to capture the nucleation statistics. Using the high-throughput approach and the appropriate experimental design to circumvent the interplay of the complicated phase segregation in membranes induced by SMase, we found that the nucleation rate of the feature can be tuned by the supersaturation of the enzyme, the lipid substrate, and the lipid product, in the fluid phase of the membrane. The correlation between the supersaturation and the nucleation rate can be well described by the classical nucleation theory equation, suggesting that the feature formation follows the nucleation process with a certain component ratio specified in the equation. The certain relative component ratio suggests that the feature may have certain organization instead of being random aggregation. In addition, our finding suggests that nucleation could serve as a time lag control mechanism in this enzymatic system, and ways to reduce nucleation energy barrier could be used to shorten the aggregation time lag and vice versa.