title: Evolutionary emergence and diversification of homo-oligomeric protein complexes
title: Evolutionäre Entstehung und Diversifizierung homo-oligomerer Proteinkomplexe
titleType: AlternativeTitle
Creators: name: Sendker, Franziska Luisa
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: protein complex
Molekularbiologie
Proteinevolution
Multiproteinkomplex
Fraktal
protein evolution
Proteine
fractal
Evolution
neutrale Evolution
Proteinkomplex
neutral theory of evolution
Chemistry &amp; allied sciences
Chemie

description: The majority of proteins do not operate in isolation but as complexes built from multiple identical copies encoded by the same gene. These homo-oligomeric complexes are ubiquitously encountered in Archaea, Bacteria and Eukaryotes and fulfil the essential functions of cellular life. Their prevalence in nature has been known for long times but the underlying causes for their emergence in evolution is less understood. Based on a frequently-held perspective that emphasizes natural selection as the most important force in evolution, they are often assumed to be a consequence of functional adaptation. While generally plausible functional benefits have been suggested for the formation of higher order assemblies, scientific evidence for specific adaptive benefits are for the most part absent. In addition, variable forms of homo-oligomeric assembly are often detected in different species while in principle performing the same function. These observations challenge an exclusively adaptive explanation for this ubiquitous form of molecular complexity. More recent theoretical studies have proposed that multimeric complexes could arise from neutral evolutionary processes and be fixed in a population by random genetic drift. This would allow proteins to sample different homo-oligomeric states over the course of evolution without an adaptive function that is selected on.
The difficulty of addressing this question has been based on an insufficient throughput and reliability of the methods that infer the homo-oligomeric state of a protein. Data that specifies how conserved or variable assembly into a distinct multimeric complex is across evolution has been very limited. In addition, deducing the effect a feature had when it first emerged in evolution is complicated as these transitions usually lie in the distant past. The subsequent accumulation of independent mutations following a transition often introduces epistasis which obscures the initial effects of evolutionary changes when investigated in modern proteins. 
In this thesis, the constraints in studying the conservation of homo-oligomeric assembly are overcome by adopting a novel experimental method – mass photometry. This technique enables the reliable and fast measurement of the multimeric state of a protein. In addition, phylogenetic methods are applied that allow the inference of the evolutionary relationship between the examined multimeric proteins from different organisms. The historic pathway of this phenotype can therefore be retraced. The identified transitions in homo-oligomeric assembly are then studied using ancestral proteins that resemble precursors of modern proteins and are resurrected using statistically approaches. These ancient proteins enable the experimental investigation of the immediate consequences that an evolutionary transition in homo-oligomeric assembly had while avoiding subsequently evolved interdependencies. The evolution of assembly into a multimeric complex and its association with protein function can thereby be studied.
The presented approach is applied in a large-scale study that characterizes the homo-oligomeric assembly of a model protein family, the citrate synthase (CS), across dozens of species and is described in Chapter 2 of this thesis. Guided by an inferred phylogeny the distribution of multimeric assembly was elucidated across evolution and revealed remarkable structural diversity. The observed evolutionary transitions included gains and losses of assembly steps as well as a case of parallel evolution of a stoichiometry. In addition, resurrected ancestral proteins revealed that the most ancient precursors of CS assembled into a distribution of multimeric complexes. These polydisperse proteins then followed a trend towards monodisperse assembly into different distinct stoichiometries. The impact on protein function during these historic transitions in homo-oligomeric assembly was examined using in vitro enzymatic assays and in vivo complementation studies. The results suggested that in most cases no obvious adaptations were conferred. This exposed a strong contrast between enormous structural plasticity and functional conservation of CS which plausibly implies that proteins can wander relatively freely between different multimeric forms over the course of evolution. 
The phylogeny-wide characterization of homo-oligomeric assembly in the CS protein family lead to the discovery of one of the most intricate examples of protein self-assembly that has been described until now. The CS from the cyanobacterium S. elongatus was found to assemble into structures that are highly reminiscent of a famous fractal pattern – the Sierpiński-triangle. The details of its assembly mechanism as well as the functional impact and evolutionary origin are extensively described in Chapter 3. Solving of a high-resolution structure via Cryo-EM allowed to unravel the intricate assembly mechanism which exploits the conformational flexibility of subcomplexes to overcome symmetrical constraints of regular closed protein assemblies. Inferred ancestral proteins pinpointed the evolution of the fractal-like assembly to a single historical substitution causing its initial emergence from simpler precursors. The in vitro functional analysis revealed that the formation of fractal-like complexes is connected to a significant reduction in enzymatic turnover. However, the performed in vivo studies in the cyanobacterial host organism did not find deleterious effects on fitness, if fractal-assembly is prevented. This suggested that a neutral evolutionary origin should even be considered for the most intricate forms of multimeric assembly and that obvious regulatory mechanisms observed in vitro are not necessarily exploited by an organism. 
Lastly, the experimental findings of plausible neutral transitions in homo-oligomeric assembly are extended to general mechanisms that can preserve such forms of molecular complexity over extended evolutionary times. Chapter 4 describes the evolutionary pathways that can drive additional molecular complexity to become completely essential. A common framework is introduced that characterizes the steps from an initially auxiliary component to becoming completely indispensable. The discussed examples include homo-oligomeric protein complexes but are also extended to heteromeric complexes, chaperones and complexes built from paralogs. It additionally addresses the erroneous inferences that are made when studying components that have become partially or completely essential by these non-adaptive mechanisms and proposes alternative procedures to circumvent these.