SpeakerProf. Victor Munoz, Host: Kevin Plaxco
Date and LocationWednesday November 12, 2014 11:00am
Proteins carry out most cellular functions, including chemical catalysis, transport, structural scaffolding, energy production, signaling, defense, and replication. To perform such roles, proteins operate as true nanomachines that rely on their ability to spontaneously self assemble onto 3D structures and use thermal and chemical energy to change shape and function. Learning how to engineer protein macromolecular assemblies would thus open a wide avenue of exciting opportunities for developing nanotechnology approaches capable of mimicking and improving nature. Interestingly, inspection of the catalog of natural protein assemblies reveals a hierarchical organization in which single-domain monomeric proteins constitute the building blocks that are successively converted onto either multi-domain proteins or assemblies of monomers. This hierarchical architecture recapitulates the course of evolution in which the interplay of genetic drift, recombination and gene duplication facilitated the progressive emergence of increasingly sophisticated assemblies. From an engineering standpoint the challenge is to define procedures that allow for the transformation of naturally monomeric proteins onto assembly-prone species and their controlled assembly to form complexes of specific size and symmetry (as opposed to non-specific protein aggregates).
As way to undertake this challenge, we have devised a simple engineering strategy that borrows ideas from molecular evolution. Particularly, we use “domain swapping”, a process by which protein molecules partially unfold to exchange one of its structural domains with in kind partners, as basic mechanism for inducing specific macromolecular assemblies. The beauty of domain swapping is that it is, in principle, general because it uses the same interaction surfaces that are already present in the folded monomer. To increase domain-swapping propensity in the monomer, we re-engineer its amino-acid sequence with the goal of decreasing its folding cooperativity. This we do either by simplifying its composition to make it more akin pre-biotic proteins, or by targeted partial deletion. In a final step, we trigger the assembly process by manipulating the stability of the folded structure (e.g. changing temperature) and/or protein concentration. Therefore, this scheme provides a built-in mechanism for controlling formation and dissociation of the macromolecular complex on demand. As proof of concept, we applied this engineering strategy to the chymotrypsin inhibitor 2 (CI2), a monomeric, superstable protein widely used as paradigmatic model of cooperative two-state folding. Our results on CI2 demonstrate the feasibility of the approach and suggest it might be generalizable.