Engineering protein activity into off-the-shelf DNA devices

Sekhon, Harsimranjit; Loh, Stewart N.

doi:10.1016/j.crmeth.2022.100202

Cited by 7 publications

(7 citation statements)

References 62 publications

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“…For this, we designed a plasmid construct expressing the picSm fused with mGL at the N-terminal for monitoring expression levels of the protein and another expressing the picLg. Additionally, we generated a construct expressing mGL-picSm and picLg fused with the 19 residue long GCN4 leucine zipper peptide that forms a parallel, two-stranded coiled-coil structure 48, 49 and has been utilized for synthetic dimerization applications including in BRET-based assays 25 . The latter two constructs were named mGL-picSm-GCN4 and picLg-GCN4, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…10, 11, 12, 13, 14, 15 Additionally, significant efforts have been made towards developing luciferases that show enhanced bioluminescence activity. These include variants that show an increased bioluminescence such as the consensus-based analysis, protein-protein interaction analysis and protease activity sensors 21, 23, 24 , including its application in protein fragment complementation assay for monitoring protein-protein interaction 25, 26, 27…”

Section: Introductionmentioning

confidence: 99%

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A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

Biswas

2023

Preprint

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Recently, a miniaturized variant of an artificial luciferase (ALuc), named picALuc, with a molecular weight of 13 kDa and thus, the smallest luciferase, was reported. While picALuc was found to be as active as the ALuc, questions remained on the structural organization and residue-residue interactions in the protein. Here, combining structural modeling, molecular dynamics (MD) simulations and mutational analysis, we show that the loss of a salt bridge interaction formed by Glu50 (E50) residue results in an increased enzymatic activity of picALuc. Specifically, we generated a model of picALuc using the available structure of the Gaussia luciferase (GLuc) and performed a 1 μs long Gaussian accelerated molecular dynamics (GaMD) simulation which revealed a general compaction of the protein structure as well as residue level interactions in the protein. Given that picALuc contains a number of charged residues, we focused our attention to salt bridge interactions and decided to mutate E10, E50 and D94 that were found to form a fluctuating, stable or a new salt bridge interaction, respectively. Live cell assays showed an enhanced bioluminescence in cells expressing the E50A mutant picALuc while in vitro assays revealed an increased Vmax of the E50A mutant without affecting its thermal stability. Dynamic cross-correlation and principal component analyses of the GaMD simulation trajectories revealed altered collective dynamics in the protein, in which residue E50 contributed substantially. Finally, we developed a protein fragment complementation assay using picALuc that allows monitoring protein-protein interaction in live cells. We envisage that the brighter variant of picALuc and the protein fragment complementation assay reported here will find a general applicability in developing bioluminescence-based assays and the strategy developed here will pave the way for further engineering of brighter variants of picALuc.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

Biswas

2023

Preprint

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“…Another area of research where NLuc has been utilized extensively is in the development of protein fragment complementation assay, typically utilized for monitoring protein-protein interaction in living cells. 24-26 Additional applications of NLuc include nanoparticle conjugates utilized for in vivo imaging 27 , sensitive viral infection detection 28 , to evaluate one of metabolic intermediate in fatty acid metabolism 29 or to test antibiotic susceptibility 30 . Further, NLuc has been successfully applied in the development of various Bioluminescence Resonance Energy Transfer (BRET)-based biomolecular assays 4-7, 31-33 such as to study molecular/organelle interactions 34, 35 , for optogenetics purposes 36 , for drug descovery 37, 38 , as genetic encoded light source for a photodynamic anticancer therapy 39 , biomolecule detection 40 , monitoring molecular tension 41 , and assays to monitor SARS-CoV-2 protease activity 42, 43 .…”

Section: Introductionmentioning

confidence: 99%

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

Ahmed

Geethakumari

Sultana

et al. 2023

Preprint

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NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

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“…In BRET-based sensor proteins, analyte binding induces a conformational change that modulates the distance and thus affects the efficiency of energy transfer between a NanoLuc donor and a fluorescent acceptor. An important advantage of these BRET-based sensor proteins is that they allow for ratiometric detection, which renders them independent of sensor concentration and much less sensitive to substrate depletion and differences in environmental conditions . However, developing sensors with a large change in BRET, and thus a high sensitivity, can be challenging as it requires conformational control of both the OFF- and ON-state of the sensor.…”

mentioning

confidence: 99%

“…An important advantage of these BRET-based sensor proteins is that they allow for ratiometric detection, which renders them independent of sensor concentration and much less sensitive to substrate depletion and differences in environmental conditions. 7 However, developing sensors with a large change in BRET, and thus a high sensitivity, can be challenging as it requires conformational control of both the OFF- and ON-state of the sensor.…”

mentioning

confidence: 99%

Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors

Gräwe

Merkx

2022

ACS Sens.

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Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5–13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

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Engineering protein activity into off-the-shelf DNA devices

Cited by 7 publications

References 62 publications

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

A Brighter picALuc Generated Through the Loss of a Salt Bridge Interaction

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors

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