How to Build a Complex, Functional Propeller Protein, From Parts.

Area of Science:

• Protein engineering
• Molecular evolution
• Biochemistry

Background:

• Proteins often evolve through the assembly of smaller functional units called motifs.
• Understanding the evolutionary pathways of complex protein structures is crucial for protein design.

Purpose of the Study:

• To investigate the evolutionary assembly of a five-bladed beta-propeller protein structure.
• To identify the ancestral sequence and evolutionary route for a single-chain fusion protein.

Main Methods:

• Ancestral sequence reconstruction (ASR) to infer ancestral protein states.
• In vitro evolution techniques to explore functional diversification.
• Analysis of protein stability and aggregation propensity in vivo.

Main Results:

• Identified individual motifs that self-assemble into a functional five-bladed beta-propeller.
• Proposed a likely evolutionary pathway from simpler motifs to a complex single-chain fusion protein.
• Observed that sequence diversification, while destabilizing some protein structures, also reduced aggregation-prone intermediates, increasing functional protein yield.

Conclusions:

• The study elucidates a plausible evolutionary trajectory for complex protein structures.
• ASR and in vitro evolution are powerful tools for dissecting protein assembly and function.
• Protein diversification can enhance functional protein levels by mitigating aggregation.