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

Biswas, Kabir H.

doi:10.1101/2023.02.14.528398

Cited by 3 publications

(1 citation statement)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When the mutations to basic residues at this position were applied to the recently developed picALuc2.0 with high bioluminescence, picALuc2.0‐E50K exhibited higher luminescence intensity than picALuc2.0‐E50R (and much higher than the original picALuc1.0). Biswas has published similar results regarding the increased bioluminescence activity of E50A in picALuc in bioRxiv, supporting our results [30]. Furthermore, in the study by Takatsu et al .…”

Section: Discussionsupporting

confidence: 91%

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Ohmuro‐Matsuyama,

Matsui,

Kanai

et al. 2023

The FEBS Journal

View full text Add to dashboard Cite

Luciferases are widely used as reporter proteins in various fields. Recently, we developed a minimal bright luciferase, picALuc, via partial deletion of the artificial luciferase (ALuc) derived from copepods luciferases. However, the structures of copepod luciferases in the substrate‐bound state remain unknown. Moreover, as suggested by structural modeling, picALuc has a larger active site cavity, unlike that in other copepod luciferases. Here, to explore the bioluminescence mechanism of picALuc and its luminescence properties, we conducted multiple mutational analyses, and identified residues and regions important for catalysis and bioluminescence. Mutations of residues likely involved in catalysis (S33, H34, and D55) markedly reduced bioluminescence, whereas that of residue (E50) (near the substrate in the structural model) enhanced luminescence intensity. Furthermore, deletion mutants (Δ70–Δ78) in the loop region (around I73) exhibited longer luminescence lifetimes (~30 min), and were reactivated multiple times upon re‐addition of the substrate. Due to the high thermostability of picALuc, one of its representative mutant (Δ74), was able to be reused, i.e., luminescence recycling, for day‐scale time at room temperature. These findings provide important insights regarding picALuc bioluminescence mechanism and copepod luciferases, and may help with sustained observations in a variety of applications.

show abstract

Section: Discussionsupporting

confidence: 91%

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Ohmuro‐Matsuyama,

Matsui,

Kanai

et al. 2023

The FEBS Journal

View full text Add to dashboard Cite

show abstract

Engineering β-catenin-derived peptides for α-catenin binding

Uddin,

Rasool,

Geethakumari

et al. 2024

emergent mater.

View full text Add to dashboard Cite

The complex formed by the β-catenin and α-catenin adaptor proteins acts as a molecular bridge that enables E-cadherin-based cell–cell adhesion assembly and maintenance in the epithelial tissue. This occurs through the interaction between the intracellular domain of E-cadherin and β-catenin on the one hand and between F-actin and α-catenin on the other hand. In addition to its role in cell–cell adhesion formation, it has been reported that E-cadherin mediates breast cancer cell metastasis to distant organs. Therefore, development of biomaterials such as peptides with ability to modulate the interaction between β-catenin and α-catenin presents an opportunity to modulate cell–cell adhesion. Here, we have performed computational and experimental analysis to develop β-catenin-derived peptides with the ability to bind α-catenin. Specifically, we analyzed the available β- and α-catenin complex structure and identified residues on β-catenin having potential to form new interactions upon mutation. We tested the wild-type (WT) and mutant β-catenin-derived peptides for their binding to α-catenin using conventional and steered molecular dynamics simulations, revealing an increased interaction of P128E and M131E mutant peptides. We then designed a Bioluminescence Resonance Energy Transfer (BRET)-based assay to monitor binding of the β-catenin-derived peptides with α-catenin, which revealed similar binding affinities of the WT and mutant β-catenin-derived peptides. Further, expression of the WT and the M131E mutant peptide resulted in a change in the aspect ratio of the cells suggestive of their ability to affect cell–cell adhesion. We envisage that the β-catenin-derived peptides engineered here will find application in blocking the interaction between β-catenin and α-catenin and, thus, modulate E-cadherin adhesion, which may lead to potential therapeutic avenue in abrogating E-cadherin-mediated metastasis of invasive breast cancer cells.

show abstract

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ahmed,

Geethakumari,

Sultana

et al. 2024

Preprint

View full text Add to dashboard Cite

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

show abstract

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

Cited by 3 publications

References 71 publications

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Glow‐type conversion and characterization of a minimal luciferase via mutational analyses

Engineering β-catenin-derived peptides for α-catenin binding

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Contact Info

Product

Resources

About