No abstract

Ordered supramolecular assemblies have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly of oppositely supercharged proteins are not fully understood. Here, we use a combination of experiments and computational modeling to systematically study the supramolecular assembly process for a series of oppositely supercharged green fluorescent protein variants. We show that net charge is a sufficient molecular descriptor to predict the interaction fate of oppositely charged proteins under a given set of solution conditions (e.g., ionic strength), but the assembled supramolecular structures critically depend on surface charge distributions. Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues not directly involved in interprotein interactions contribute to a substantial fraction (∼30%) of the interaction energy between oppositely charged proteins via long-range electrostatic interactions. Dynamic subunit exchange experiments further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of ∼10–40 min, which is governed by protein composition and solution conditions. Broadly, our results inform how protein supercharging can be used to create different ordered supramolecular assemblies from a single parent protein building block.

Ordered supramolecular assemblies of supercharged synthetic proteins have recently been created using electrostatic interactions between oppositely charged proteins. Despite recent progress, the fundamental mechanisms governing the assembly process between oppositely supercharged proteins are not fully understood. In this work, we use a combination of experiments and computational modeling to systematically study the supramolecular assembly process for a series of oppositely supercharged green fluorescent protein (GFP) variants. Our results show that the assembled structures of oppositely supercharged proteins critically depend on surface charge distributions. In addition, net charge is a sufficient molecular descriptor to predict the interaction fate of oppositely charged proteins under a given set of solution conditions (e.g., ionic strength). Interestingly, our results show that a large excess of charge is necessary to nucleate assembly and that charged residues that are not directly involved in interprotein interactions contribute to a substantial fraction (~30%) of the interaction energy between oppositely charged proteins via long-range electrostatic interactions. Dynamic subunit exchange experiments enabled by Forster resonance energy transfer (FRET) further show that relatively small, 16-subunit assemblies of oppositely charged proteins have kinetic lifetimes on the order of ~10-40 minutes, which is governed by protein composition and solution conditions. Overall, our work shows that a balance between kinetic stability and electrostatic charge ultimately determine the fate of supramolecular assemblies of supercharged proteins. Broadly, our results inform how protein supercharging can be used to generate different ordered supramolecular assemblies from a single parent protein building block.

No abstract

Smoothened (SMO) is a membrane protein of the Class F subfamily of G-Protein Coupled Receptors (GPCRs) and maintains homeostasis of cellular differentiation. SMO undergoes conformational change during activation, transmitting the signal across the membrane, making it amenable to bind to its intracellular signaling partner. Receptor activation has been studied at length for Class A receptors, but the mechanism of Class F receptor activation remain unknown. SMO bound to agonists and antagonists at sites in the Transmembrane Domain (TMD) and the Cysteine Rich Domain (CRD) has been characterized; giving a static view of the various conformations SMO adopts. While these crystal structures of the inactive and active SMO outline the residue-level transitions, a kinetic view of the overall activation process remains unexplored for Class F receptors. We describe SMO's activation process in atomistic detail by performing 300 micro-seconds of molecular dynamics simulations and combining it with Markov State Model theory. A molecular switch, conserved across Class F and analogous to the activation-mediating D-R-Y motif in Class A receptors, is observed to break during activation. We also show that this transition occurs in a stage-wise movement of the transmembrane helices - TM6 first, followed by TM5. To see how modulators affect SMO activity, we simulated agonist and antagonist-bound SMO. We observed that agonist-bound SMO has an expanded hydrophobic tunnel in SMO's core TMD, while antagonist-bound SMO shrinks this tunnel; further supporting the hypothesis that cholesterol travels through a tunnel inside Smoothened to activate it. In summary, this study establishes the distinct activation mechanism of Class F GPCRs and shows that SMO's activation process rearranges the core transmembrane domain to open a hydrophobic conduit for cholesterol transport.