Calcium-responsive order-to-order transitions by repetitive bacterial proteins

16 May 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Protein sequences dictate their functions, and sequence repetition is a common strategy to amplify function within modular, protein-based biomaterials and biotechnologies. Repetitive bacterial proteins have drawn interest for their 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 bacterial protein that forms β-roll structures in the presence of calcium. Sequence modifications focused on the repetitive region, including either global substitution of nonconserved residues or complete replacement with tandem repeats of the consensus nonapeptide GGAGXDTLY. Some sequence modifications disrupted the disorder-to-order transition associated with β-roll formation, despite conservation of the underlying nonapeptide sequence. Proteins enriched in smaller, hydrophobic amino acids adopted secondary structures in the absence of calcium and underwent order-to-order structural transitions 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 residues in calcium-responsive folding, thereby revealing a strategy to leverage these residues in the design of tunable, calcium-responsive biomaterials.

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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