FUNCTION-DRIVEN APPROACHES TO THE DESIGN OF OPTOGENETIC TOOLS

Abstract

Proteins play a wide variety of roles in biology despite being produced from a small set of common subunits; this commonality can be exploited to understand the dynamics by which proteins fold into structures and perform their manifold functions and, subsequently, design new proteins for use both in research and as nanoscale machines in industry. While this design process has classically involved residue-level redesign of existing protein backbones and, more recently, the de novo design of backbones according to geometrical parameters, the increasing complexity of optogenetic photosystems, biosensors, and other mechanisms for making use of proteins with specific functions has established a need for a design protocol that can reconcile their various structural exigencies with the function-specific elements of as wide an array of proteins as possible in order to make best use of them. Requirement-driven design eschews specific structural templates in favor of general requirements dependent on the intended function of the design, and so can exploit the vastness of protein structural space in finding solutions to increasingly complex design problems. Here, we present three new advances in the requirement-driven design of proteins as diagnostic tools, including a more general photosystem for the direct optogenetic control of protein-protein interactions, a series of algorithmic improvements to the leading implementation of requirement-driven design in the Rosetta macromolecular design software suite, and a new version of that algorithm capable of performing requirement-driven backbone design and residue-level backbone optimization simultaneously. These technologies collectively represent a significant improvement in our ability to control the activity of proteins with a wide variety of control schemes and produce functional proteins for arbitrary requirement sets more generally.Doctor of Philosoph