Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries

Ahmed, Shahbaz; Manjunath, Kavyashree; Chattopadhyay, Gopinath; Varadarajan, Raghavan

doi:10.1016/j.jbc.2022.101785

Cited by 15 publications

(36 citation statements)

References 58 publications

(98 reference statements)

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“…The global suppressor R10G rescued GyrA14-binding defects at all five PIM positions, and increased the apparent T m by 8 °C, relative to WT CcdB (1) but E11R was not characterized. Subsequently, another putative global suppressor S12G was identified by analyzing saturation suppressor libraries using FACS coupled to deep sequencing (13). S12, located beside R10 and E11 on the exposed loop, is involved in hydrogen bonding with the cognate antitoxin CcdA (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…In order to further, investigate the role of suppressors on protein stability in the WT background, we performed detailed thermodynamic and kinetic studies of the suppressors alone in CcdB and mRBD proteins (Figure 5). In a recent study employing the PIMs L36A, V18D, V18G and V20G (chosen to span a range of stabilities), several other CcdB suppressor substitutions were also identified using yeast surface display coupled to deep sequencing (13). In the present study, we selected three such suppressor substitutions, Y8D, V46L and S60E with ΔT m ˃3 °C (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

“…From our previous studies that have characterised a large number of CcdB mutants by YSD, L42E and S43T were seen to exhibit higher binding than WT and were presumed to be more stable (36). However, these mutants were not identified as suppressors using YSD (13). In the same study, M32T was identified as a suppressor (13).…”

Section: Resultsmentioning

confidence: 99%

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Mechanistic insights into global suppressors of protein folding defects

Chattopadhyay

Bhowmick

Manjunath

et al. 2021

Preprint

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Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined five global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate parameters. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. Our study also demonstrates that the global suppressor approach can be used to consistently stabilise wild-type proteins, including for downstream translational applications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Mechanistic insights into global suppressors of protein folding defects

Chattopadhyay

Bhowmick

Manjunath

et al. 2021

Preprint

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“…The methodology was used to provide insights into the rejuvenation process and identify mutations that stabilized otherwise highly transient species. In this proof of principle study, down selected mutants were individually cloned and characterized, but in future, mutant pools isolated by FACS can be analysed by deep sequencing to further enhance throughput (Adams et al, 2016; Ahmed et al, 2022b, 2022a; Noderer et al, 2014; Reich et al, 2015; Starr et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…The SPR experiments have been performed once with four different concentrations of the protein (in each experiment) and the listed error is the standard error derived from the values at multiple concentrations. a Parameters from previous study(Ahmed et al, 2022;.…”

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confidence: 99%

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Chattopadhyay

Ahmed

Srilatha

et al. 2022

Preprint

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Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterisation is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a YSD saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used SPR with purified proteins to validate the kinetics measured using surface display. Positions where CcdB mutations lead to slower rejuvenation rates are largely involved in CcdA-binding, though there were several notable exceptions. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring kinetics of ternary complex formation for individual CcdB mutants in solution by FRET studies.

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Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

et al. 2022

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Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.

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Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries

Cited by 15 publications

References 58 publications

Mechanistic insights into global suppressors of protein folding defects

Mechanistic insights into global suppressors of protein folding defects

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Ter‐Seq: A high‐throughput method to stabilize transient ternary complexes and measure associated kinetics

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