Methyl-ß-cyclodextrin asymmetrically extracts phospholipid from bilayers, granting tunable control over differential stress in lipid vesicles.

Lipid asymmetry - that is, a nonuniform lipid distribution between the leaflets of a bilayer - is a ubiquitous feature of biomembranes and is implicated in several cellular phenomena. Differential tension - that is, unequal lateral monolayer tensions comparing the leaflets of a bilayer- is closely associated with lipid asymmetry underlying these varied roles. Because differential tension is not directly measurable in combination with the fact that common methods to adjust this quantity grant only semi-quantitative control over it, a detailed understanding of lipid asymmetry and differential tension are impeded. To overcome these challenges, we leveraged reversible complexation of phospholipid by methyl-ß-cyclodextrin (mbCD) to tune the direction and magnitude of lipid asymmetry in synthetic vesicles. Lipid asymmetry generated in our study induced (i) vesicle shape changes and (ii) gel-liquid phase coexistence in 1-component vesicles. By applying mass-action considerations to interpret our findings, we discuss how this approach provides access to phospholipid thermodynamic potentials in bilayers containing lipid asymmetry (which are coupled to the differential tension of a bilayer). Because lipid asymmetry yielded by our approach is (i) tunable and (ii) maintained over minute to hour timescales, we anticipate that this approach will be a valuable addition to the experimental toolbox for systematic investigation into the biophysical role(s) of lipid asymmetry (and differential tension).

Affinity Bioorthogonal Chemistry (ABC) Tags for Site-Selective Conjugation, On-Resin Protein-Protein Coupling, and Purification of Protein Conjugates.

The site-selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl-tetrazines, the nickel-iminodiacetate (Ni-IDA) resins commonly used for His-tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity-based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC-tagging works with a range of site-selective bioconjugation methods with proteins tagged at the C-terminus, N-terminus or at internal positions. ABC-tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site-selective conjugation and clean-up with ABC-tagged proteins also allows for facile on-resin reactions to provide protein-protein conjugates.