Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins

Perez-Riba, Albert; Lowe, Alan R.; Main, Ewan R. G.; Itzhaki, Laura S.

doi:10.1016/j.bpj.2018.03.038

Cited by 10 publications

(36 citation statements)

References 55 publications

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“…These studies will allow us to understand the relationship between the length/sequence composition of the grafted peptide and its binding affinity within the context of the CTPR scaffold. In terms of the maximum peptide length, we have found that we can successfully graft binding peptides of up to 15 residues onto the inter‐repeat loop, and we have also shown that the loop can be extended by up to 40 residues without disrupting the CTPR structure (; unpublished results).…”

Section: Resultsmentioning

confidence: 79%

“…The high thermodynamic stabilities of CTPRs combined with their modular nature means that even gross modifications are still likely to produce proteins with high stabilities, and indeed we recently showed that CTPR proteins can tolerate extension of the inter‐repeat loop by up to 25 residues . Here, we take advantage of this malleability to describe a new strategy for engineering high‐affinity binding functions into CTPR proteins by grafting functional peptides into the inter‐repeat loop.…”

Section: Introductionmentioning

confidence: 99%

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Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

2019

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Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra-stable consensus-designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch-like ECH-associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1-binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.

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Section: Resultsmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

2019

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“…This may not be the case for other proteins and has to be verified on a case-by-case basis. Moreover, the application of the ybbR-tag has limitations for the introduction of internal attachments sites, particularly in a repeat protein context where we have found that simple unstructured loops destabilize the array 23 . We expect that the introduction of a short α-helix would have an even greater effect, even if it is accompanied by unstructured spacers either side.…”

Section: Resultsmentioning

confidence: 95%

Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Synakewicz

Bauer

Rief

et al. 2019

Sci Rep

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Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.

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“…This is referred to as a homopolymer Ising model 10,25 . The stabilities of non-identical repeat proteins can also be described using an Ising model, but a more complex description is required, which is referred to as the heteropolymer model 25–29 .…”

Section: Introductionmentioning

confidence: 99%

Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Perez-Riba

Komives

Main

et al. 2019

Sci Rep

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The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

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Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins

Cited by 10 publications

References 55 publications

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

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