Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials

Adam J Wargacki,Tobias P Wörner,Neil P King,Albert J R Heck,Michiel Van De Waterbeemd,Daniel Ellis

doi:10.1038/s41467-021-21251-y

Abstract

Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.

Highlights

Recent advances in computational methods have enabled the predictive design of selfassembling protein nanomaterials with atomic-level accuracy
Non-cooperative folding of de novo designed proteins[39], along with parallels drawn between cooperative virus capsid assembly and two-state intramolecular folding[21], led us to investigate the assembly of computationally designed protein nanomaterials that lack an evolutionary history
We observe that cooperative assembly can be intrinsic to hierarchically structured protein nanomaterials, but the noncooperative assembly observed for I53-50 in certain regimes indicates it is a conditional rather than a universal feature

Summary

Introduction

Recent advances in computational methods have enabled the predictive design of selfassembling protein nanomaterials with atomic-level accuracy These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. Many proteins assemble into multi-subunit complexes in order to perform highly specialized functions The sophistication of these molecular machines has inspired efforts to adapt them to new purposes by making small alterations through mutation. Two strategies have repeatedly achieved predictive positioning of protein subunits in three-dimensional complexes with atomic-level accuracy: the genetic fusion method[3,4] and computational docking followed by protein– protein interface design[5,6,7,8,9,10] This ability has opened up the possibility of tailoring the structures of multi-subunit complexes to specific functions, a long-standing goal in biotechnology. Our detailed characterization of computationally designed protein complexes lacking an evolutionary history reveals aspects of protein assembly pathways that are intrinsic to stable multi-subunit complexes[39]

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Conclusion