Benchmarking cassette-based deep mutagenesis by Golden Gate assembly

Daffern, Nicolas; Baumer, Zachary T; Francino-Urdániz, Irene M.; Whitehead, Timothy A.

doi:10.1101/2023.04.13.536781

Cited by 2 publications

(2 citation statements)

References 27 publications

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“…Based on previous experience with poor prediction of electrostatic interactions by Rosetta, we excluded mutations at PYR1 positions 59 and 141, instead incorporating the PYR1 WIN -mutation K59Q into designs. DNA sequences of the wild-type PYR1 sequence with combinations of the point mutations K59Q, S92V, F159A, F159V, A160I, and A160V were ordered (Integrated DNA Technologies) and cloned by Golden Gate Assembly 51 into the pND003 vector. Constructs were expressed in yeast surface display and binding affinity to WIN55212,2 was analyzed as previously described for isogenic variants.…”

Section: Methodsmentioning

confidence: 99%

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design

Leonard,

Friedman,

Chayer

et al. 2024

Preprint

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The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem which could enable many practical applications of protein biosensors. In this work, we analyzed two engineered biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the syn-thetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complemen-tarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands. MD simula-tions indicate that both PYR1 protein-ligand complexes bind a single conformer of their target ligand that is close to the lowest free energy conformer. Computational design using a fixed conformer and rigid body orientation led to new WIN55,212-2 sensors with nanomolar limits of detection. This work reveals mechanisms by which the versatile PYR1 bio-sensor scaffold can bind diverse ligands. This work also provides computational methods to sample realistic ligand conform-ers and rigid body alignments that simplify the computational design of biosensors for novel ligands of interest.

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Section: Methodsmentioning

confidence: 99%

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design

Leonard,

Friedman,

Chayer

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Based on previous experience with poor prediction of electrostatic interactions by Rosetta, we excluded mutations at PYR1 positions 59 and 141, instead, we incorporated the PYR1 WIN -mutation K59Q into designs. DNA sequences of the wild-type PYR1 sequence with combinations of point mutations K59Q, S92 V, F159A, F159 V, A160I, and A160 V were ordered (Integrated DNA Technologies) and cloned by Golden Gate Assembly into the pND003 vector. Constructs were expressed in yeast surface display and binding affinity to WIN55212,2 was analyzed as previously described for isogenic variants.…”

Section: Methodsmentioning

confidence: 99%

Rationalizing Diverse Binding Mechanisms to the Same Protein Fold: Insights for Ligand Recognition and Biosensor Design

Leonard,

Friedman,

Chayer

et al. 2024

ACS Chem. Biol.

View full text Add to dashboard Cite

The engineering of novel protein−ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem, which could enable many practical applications of protein biosensors. In this work, we analyzed two engineered biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein−ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands. MD simulations indicate that both PYR1 protein−ligand complexes bind a single conformer of their target ligand that is close to the lowest free-energy conformer. Computational design using a fixed conformer and rigid body orientation led to new WIN55,212-2 sensors with nanomolar limits of detection. This work reveals mechanisms by which the versatile PYR1 biosensor scaffold can bind diverse ligands. This work also provides computational methods to sample realistic ligand conformers and rigid body alignments that simplify the computational design of biosensors for novel ligands of interest.

show abstract

Benchmarking cassette-based deep mutagenesis by Golden Gate assembly

Cited by 2 publications

References 27 publications

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design

Rationalizing Diverse Binding Mechanisms to the Same Protein Fold: Insights for Ligand Recognition and Biosensor Design

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