The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

Evans, Geraint; Craggs, Timothy D.; Kapanidis, Achillefs N.

doi:10.1016/j.jmb.2021.167410

Cited by 6 publications

(1 citation statement)

References 42 publications

(95 reference statements)

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“…Experimentally, several conformational states have been observed by x-ray crystallography and cryo-EM (Verba et al 2016;Ali et al 2006;Bron et al 2008). The dynamics between these conformational states can be measured with single-molecule Förster Resonance Energy Transfer (smFRET), similar to many other protein systems (Tassis et al 2021;Evans et al 2022;Krainer et al 2020;Steffen et al 2020).…”

Section: 𝜋 𝑖 𝑝 𝑖𝑗 = 𝜋 𝑗 𝑝 𝑗𝑖mentioning

confidence: 99%

Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90

Vollmar

Schimpf

Hermann

et al. 2023

Preprint

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The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90's conformational changes during its interaction with the client kinase Ste11. Most interestingly, three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation.

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Section: 𝜋 𝑖 𝑝 𝑖𝑗 = 𝜋 𝑗 𝑝 𝑗𝑖mentioning

confidence: 99%

Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90

Vollmar

Schimpf

Hermann

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Strategies and procedures to generate chimeric DNA polymerases for improved applications

Yu,

Wang

2024

Appl Microbiol Biotechnol

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Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. Key points • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.

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Awakening a latent phosphoenolpyruvate- oxaloacetate-glyceraldehyde carbon-fixation pathway for cost-effective nitrogen removal by adjusting carbon source and pH in the anammox-centered process

Hao,

Zhang,

Yang

et al. 2024

Chemical Engineering Journal

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The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

Abstract: Graphical abstract

Cited by 6 publications

References 42 publications

Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90

Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90

Strategies and procedures to generate chimeric DNA polymerases for improved applications

Awakening a latent phosphoenolpyruvate- oxaloacetate-glyceraldehyde carbon-fixation pathway for cost-effective nitrogen removal by adjusting carbon source and pH in the anammox-centered process

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