Diffusion-induced competitive two-site binding

Gopich, Irina V.; Szabó, Attila

doi:10.1063/1.5079748

Cited by 13 publications

(10 citation statements)

References 28 publications

(48 reference statements)

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Section: Resultsmentioning

confidence: 99%

“…22 However, this modest drop in rate constant is not necessarily indicative of the encapsulin shell as a rate-determining barrier to diffusion, as for example, competition between multiple encapsulated LBTs within the encapsulin lumen could also result in complex kinetics that can lower the apparent first-order rate. 47,48 Furthermore, all pore variants exhibited relatively similar slow rate constants for Tb 3+ turn-on kinetics (Figure 4c, SI Figure S7.1), suggesting that the overall kinetics may primarily be governed by Tb 3+ -LBT luminescent complex formation as a rate-determining step, rather than transit through the pores. Apart from S3E and S6E which showed slight increases over the wild-type rate with pseudo-first order rate constants of 55±3 s -1 and 70±4 s -1 respectively, there were minimal differences in rate for the remainder of the variants, including the S7G control featuring a wide but positively-charged five-fold pore (46±2 s -1 ), which had previously been reported to give a seven-fold increase in Tb 3+ flux using a comparable experimental setup.…”

Section: Overall Reaction Kinetics Depends On Whether Influx or Cargo Kinetics Is Rate Determiningmentioning

confidence: 94%

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Pore structure controls stability and molecular flux in engineered protein cages

Adamson

Tasneem

Andreas

et al. 2021

Preprint

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Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. Pores lined with anionic residues resulted in more robust assemblies than their corresponding cationic variants. We then determined seven cryo-EM structures of pore variants at resolutions between 2.5-3.6 Å. Together with stopped-flow kinetics experiments for quantifying cation influx, we uncover the complex interplay between pore size, charge, and flexibility that controls molecular flux in protein cages, providing guidance for future nanoreactor designs.Abstract Figure

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Section: Resultsmentioning

confidence: 99%

Section: Overall Reaction Kinetics Depends On Whether Influx or Cargo Kinetics Is Rate Determiningmentioning

confidence: 94%

Pore structure controls stability and molecular flux in engineered protein cages

Adamson

Tasneem

Andreas

et al. 2021

Preprint

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“…The effective affinity is likely to be raised through both an increase in the number of productive encounters given the high density of SLiMs, and through rebinding mechanisms that reduce the diffusion of Calcineurin away from the scaffold since the molecule is likely to be intercepted by another binding site rather than diffusing away. We note that a recently developed model for competitive two-site binding showed that the bound population at one site can influence kinetics of binding at the other where they are connected by a diffusion pathway (39). This latter point is borne out directly in our experiments by the lack of complete dissociation of Calcineurin on AKAP5c seen by SPR (Fig.…”

Section: Discussionmentioning

confidence: 99%

Hidden multivalency in phosphatase recruitment by a disordered AKAP scaffold

Watson¹,

Almeida²,

A³

et al. 2021

Preprint

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Signalling requires precise spatial and temporal regulation of molecular interactions, which is frequently orchestrated by disordered scaffolding proteins, such as A-kinase anchoring protein 5 (AKAP5). AKAP5 contains multiple Short Linear Motifs (SLiMs) that assemble the necessary components, including the phosphatase Calcineurin, which is anchored via a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that Calcineurin also recognises additional lower-affinity SLiMs C-terminal to the PxIxIT motif. Moreover, we demonstrate that the assembly is in reality a complex system in which AKAP SLiMs spanning a wide affinity range act cooperatively to maintain distinct pools of anchored and more loosely held enzyme, analogous to the well-understood transcription factor search complexes on DNA, and compatible with the requirement for both stable anchoring and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that cooperate to maintain a structurally disordered but tightly regulated signalosome.

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“…Recently, in an article, Szabo et al investigated the effect of ligand diffusivity on the reversible binding kinetics of a ligand to a macromolecule with two distinct competitive binding sites. In the fast diffusion limit, the two sites behave quite independentlyoccupancy of a ligand to one site is independent of the other; thus, the reaction network follows the ordinary chemical kinetics.…”

Section: Introductionmentioning

confidence: 99%

“…We present the rate equations associated with reversible ligand binding to a macromolecule with two competitive binding sites within Markovian Wilemski–Fixman approximation in Section of the Methods section. This sub-section is basically a recapitulation of Szabo et al’s paper, which we need to keep for the sake of completeness. The effects of ligand diffusion on the precision of single site occupancy, the mean dynamical activity, and the mean entropy production rate are calculated in Section .…”

Section: Introductionmentioning

confidence: 99%

Statistics of Reaction Flux and Dynamical Activity Associated with a Diffusion-Influenced Ligand-Binding Reaction

Ghosal

2021

J. Phys. Chem. B

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In this paper, we consider a macromolecule with two competitive binding sites where a ligand can bind to and gives rise to a unicyclic reaction network consisting of four states(i) a single state with both binding sites vacant, (ii) two states with one bound site and one free binding site, and (iii) an another single state with both sites occupied. We obtain probability densities of the time-integrated current along the clockwise direction and the dynamical activity or mean number of jumps between different states for finite times at a fast diffusion limit. On the other hand, in the diffusion-limited case, ligand diffusion between the two binding sites directly connects the mono-ligated stateschanging the reaction scheme. Addition of the new reaction channel alters the precision of ligand occupancy to a single site, the mean dynamical activity, and the mean entropy production rate. All of these quantities are calculated with varying degrees of competition between the two sites for ligands, and we find that increase in the competition between the two sites decreases all above-mentioned additive functionals. The upper bound of precision associated with a single-site ligand occupancy for a diffusion-influenced reaction network is set by the mean dynamical activity (mean entropy production rate) at small (large) ligand concentrations at the steady-state limit.

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Diffusion-induced competitive two-site binding

Abstract: Note: This article is part of the Special Topic "Markov Models of Molecular Kinetics" in J. Chem. Phys.

Cited by 13 publications

References 28 publications

Pore structure controls stability and molecular flux in engineered protein cages

Pore structure controls stability and molecular flux in engineered protein cages

Hidden multivalency in phosphatase recruitment by a disordered AKAP scaffold

Statistics of Reaction Flux and Dynamical Activity Associated with a Diffusion-Influenced Ligand-Binding Reaction

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