Unravelling the Evolution of Complexes with Ancestral Sequence Reconstruction

Introduction

Almost all proteins perform their functions as part of protein complexes. These assemblies are intricate and often beautiful examples of evolution's capacity to generate complexity. But are they built by natural selection? My recent work has revealed that they may in fact be produced and maintained across vast time scales by neutral processes, even if they provide no adaptive benefit at all.

Objectives

In this proposal, I will test this radical idea by bringing together ancestral sequence reconstruction and quantitative biochemistry of protein complexes. I will use this approach to experimentally unravel the evolutionary processes that generate and maintain biochemical complexity in three model systems that exemplify archetypical protein-protein interactions:

Objective 1: Homomeric Interactions

1. I will unravel what drives the evolutionary gain and loss of homomeric interactions.
2. I will recapitulate how the universally conserved enzyme citrate synthase repeatedly underwent changes in self-assembly state.
3. I will test if these changes were adaptive or whether they resulted from new interfaces being highly evolvable.

Objective 2: Transient Interactions with Chaperones

1. I will probe why protein complexes evolve to depend on transient interactions with folding and assembly chaperones.
2. I will retrace how the CO2 fixing enzyme RubisCO acquired a set of dedicated assembly and folding chaperones.
3. I will unravel whether the initial gain of new chaperone interactions was useful and determine what later caused RubisCO to start completely depending on them.

Objective 3: Neutral Evolution of Interactions

1. I will unravel if many of the interactions between different complexes are caused by neutral evolution.
2. I will use a new experimental approach in yeast to quantify the rate at which complexes gain interactions with the rest of the proteome by chance alone.

Conclusion

Together, these experiments will show for the first time how adaptive evolution and neutral processes interacted to produce the intricate biochemical complexity we see inside cells today.