Synthesis and modification of alternative copolymers for activating membrane enzymes in vitro

Poly(styrene-co-maleic acid) (SMA) is increasingly being used for the solubilization of lipid membranes and membrane proteins is becoming more widespread. To this end, a deeper understanding of the chemical properties of the copolymer and how these translate into membrane solubilization properties and improvement of enzyme activity or stability to better promote the in vitro reaction of membrane enzymes. SMA comes in several different flavors that include the ratio of styrene to maleic acid, the distribution of comonomer sequences, the average chain length, dispersity, and potential chemical modifications. We have introduced a group of SMAs whose composition and length are well defined in a stepwise manner, and obtained the polymer (4-Cl-SMA-QA) for (-)-trans-isopiperitenone dehydrogenase (IPDH) with a single-pass membrane structure domain. The protein extraction efficiency of 4-Cl-SMA-QA is higher than that of the widely used commercial SMA(2/1), up to 82%, the kcat of IPDH embedded in 4-Cl-SMA-QA is increased by a factor of 4, a 30% decrease in Km, and a 5-fold increase in overall catalytic performance. The requirements of SMA lipid particles’ size for single-span and multi-span membrane proteins are analyzed, which is helpful for rapidly selecting suitable scaffold polymers from massive polymer libraries.