Alternatives to Styrene- and Diisobutylene-Based Copolymers for Membrane Protein Solubilization via Nanodisc Formation.

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.

Effects of Esterified Styrene-Maleic Acid Copolymer Degradation on Integral Membrane Protein Extraction.

The detergent-free extraction of integral membrane proteins using styrene-maleic acid copolymers (SMAs) has shown promise as a potentially effective technique to isolate proteins in a more native-like conformation. As the field continues to develop, the protein selectivity and extraction efficiency of many analogues of traditional SMAs are being investigated. Recently, we discovered that the monoesterification of SMAs with alkoxy ethoxylate sidechains drastically affects the bioactivity of these copolymers in the extraction of photosystem I from the cyanobacterium Thermosynechococcus elongatus. However, subsequent investigations also revealed that the conditions under which these esterified SMA polymer analogues are prepared, purified, and stored can alter the structure of the alkoxy ethoxylate-functionalized SMA and perturb the protein extraction process. Herein, we demonstrate that the basic conditions required to solubilize SMA analogues may lead to deleterious saponification side reactions, cleaving the sidechains of an esterified SMA and dramatically decreasing its efficacy for protein extraction. We found that this process is highly dependent on temperature, with polymer samples being prepared and stored at lower temperatures exhibiting significantly fewer saponification side reactions. Furthermore, the effects of small-molecule impurities and exposure to light were also investigated, both of which are shown to have significant effects on the polymer structure and/or protein extraction process.

Protein Extraction Efficiency and Selectivity of Esterified Styrene-Maleic Acid Copolymers in Thylakoid Membranes.

Amphiphilic styrene-maleic acid copolymers (SMAs) have been shown to effectively extract membrane proteins surrounded by an annulus of native membrane lipids via the formation of nanodiscs. Recent reports have shown that 2-butoxyethanol-functionalized SMA derivatives promote the extraction of membrane proteins from thylakoid membranes, whereas unfunctionalized SMA is essentially ineffective. However, it is unknown how the extent of functionalization and identity of sidechains impact protein solubilization and specificity. Herein, we show that the monoesterification of an SMA polymer with hydrophobic alkoxy ethoxylate sidechains leads to an increased solubilization efficiency (SE) of trimeric photosystem I (PSI) from the membranes of cyanobacterium Thermosynechococcus elongatus. The specific SMA polymer used in this study, PRO 10235, cannot encapsulate single PSI trimers from this cyanobacterium; however, as it is functionalized with alkoxy ethoxylates of increasing alkoxy chain length, a clear increase in the trimeric PSI SE is observed. Furthermore, an exponential increase in the SE is observed when >50% of the maleic acid repeat units are monoesterified with long alkoxy ethoxylates, suggesting that the PSI extraction mechanism is highly dependent on both the number and length of the attached side chains.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.