Monitoring protein conformational changes using fluorescent nanoantennas

Harroun, Scott G.; Lauzon, Dominic; Ebert, Maximilian; Desrosiers, Arnaud; Vallée‐Bélisle, Alexis

doi:10.1038/s41592-021-01355-5

Cited by 22 publications

(30 citation statements)

References 86 publications

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“…These states interchange in harmony with changes in solvent properties, such as pH, salt, inhibitors, or chaotropic salts, and physical conditions, such as temperature. Further development of sensitive single-molecule techniques − combined with classical methods will undoubtedly link functional effects to structural alterations in APs, distinguishing between the various enzyme, enzyme–substrate, and enzyme–product forms.…”

Section: Discussionmentioning

confidence: 99%

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

et al. 2022

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Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0−1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using sitedirected mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

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Section: Discussionmentioning

confidence: 99%

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase

et al. 2022

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“…Five distinct conformational states were characterized, including the transient enzyme-substrate complex 64 .…”

Section: Discussionmentioning

confidence: 99%

“…It was concluded that the dye-enzyme interaction mediated by the nanoantenna allowed monitoring of the conformational changes on the enzyme surface during its function. Five distinct conformational states were characterized, including the transient enzyme–substrate complex 64 .…”

Section: Discussionmentioning

confidence: 99%

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Markússon

Hjörleifsson

Kursula

et al. 2022

Preprint

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Enzyme stability and function can be affected by various environmental factors, such as temperature, pH and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0 – 1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small angle X-ray scattering (SAXS) showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

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“…An exciting new tool to study conformational dynamics, recently developed by Harroun et al . ( 70 ), involves the attachment of fluorescent nanoantenna to a protein in a less functionally invasive way than traditional methods. This newly developed technique is similar to previously described methods that conjugate fluorescent dyes, such as Alexa488 and 594 to proteins, with the exception of utilizing a “nanoantenna” consisting of biotin, a linker region, and a fluorescent dye.…”

Section: Llps and Scaffold Assemblymentioning

confidence: 99%