Sequence-defined structural transitions by calcium-responsive proteins

20 August 2024, Version 3
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Biopolymer sequences dictate their functions, and protein-based polymers are a promising platform to establish sequence–function relationships for novel biopolymers. To efficiently explore vast sequence spaces of natural proteins, sequence repetition is a common strategy to tune and amplify specific functions. This strategy is applied to repeats-in-toxin (RTX) proteins with calcium-responsive folding behavior, which stems from tandem repeats of the nonapeptide GGXGXDXUX in which X can be any amino acid and U is a hydrophobic amino acid. To determine the functional range of this nonapeptide, we modified a naturally occurring RTX protein that forms β-roll structures in the presence of calcium. Sequence modifications focused on calcium-binding turns within the repetitive region, including either global substitution of nonconserved residues or complete replacement with tandem repeats of a consensus nonapeptide GGAGXDTLY. Some sequence modifications disrupted the typical transition from intrinsically disordered random coils to folded β rolls, despite conservation of the underlying nonapeptide sequence. Proteins enriched in smaller, hydrophobic amino acids adopted secondary structures in the absence of calcium and underwent structural rearrangement in calcium-rich environments. In contrast, proteins with bulkier, hydrophilic amino acids maintained intrinsic disorder in the absence of calcium. These results indicate a significant role of nonconserved amino acids in calcium-responsive folding, thereby revealing a strategy to leverage sequence in the design of tunable, calcium-responsive biopolymers.

Keywords

calcium responsive
tandem repeat proteins
consensus sequence
protein design
protein stability

Supplementary materials

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Description
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Supporting Information
Description
DNA and protein sequences for RTX variants, protein properties, detailed calculations, and circular dichroism spectroscopy replicates
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