A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed by single-molecule FRET

Abstract“CLC” transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Förster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Å) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.SUMMARYCheng, Krishnamoorti et al. use single-molecule Förster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

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Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase

Nature Communications ◽

10.1038/s41467-021-22562-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Simon Bo Jensen ◽

Sara Thodberg ◽

Shaheena Parween ◽

Matias E. Moses ◽

Cecilie C. Hansen ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Cytochromes P450 ◽

Conformational Sampling ◽

P450 Oxidoreductase ◽

Single Molecule Fret ◽

Biased Signaling ◽

Small Molecule Ligands ◽

Cell Assays ◽

And Function

AbstractMetabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

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S09A1 NMR analysis of subunit conformational change induced by ligand binding in F_1-ATPase(Mechanism of F_1-ATPase Molecular Motor-A Cross Talk among Single Molecule, Structural Biology, and Molecular Simulation Studies-)

Seibutsu Butsuri ◽

10.2142/biophys.47.s12_4 ◽

2007 ◽

Vol 47 (supplement) ◽

pp. S12

Author(s):

Hiromasa Yagi ◽

Hideo Akutsu

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Molecular Simulation ◽

Structural Biology ◽

Single Molecule ◽

Cross Talk ◽

Molecular Motor ◽

Simulation Studies ◽

Nmr Analysis

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Wavelet Shrinkage to Resolve Single Molecule FRET Structural Landscape of the Isolated Ligand Binding Domain of the AMPA Receptor

Biophysical Journal ◽

10.1016/j.bpj.2010.12.2800 ◽

2011 ◽

Vol 100 (3) ◽

pp. 478a

Author(s):

Christy F. Landes ◽

J. Nick Taylor ◽

Anu Rambhadran ◽

Vasanthi Jayaraman

Keyword(s):

Ligand Binding ◽

Ampa Receptor ◽

Single Molecule ◽

Binding Domain ◽

Ligand Binding Domain ◽

Wavelet Shrinkage ◽

Single Molecule Fret

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Activation of A1 Domain Adhesiveness in von Willebrand Factor by Elongational Force

Blood ◽

10.1182/blood.v120.21.sci-16.sci-16 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-16-SCI-16

Author(s):

Timothy A. Springer ◽

Jongseong Kim

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Von Willebrand Factor ◽

Single Molecule ◽

Conflicts Of Interest ◽

Stable State ◽

Receptor Ligand ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract SCI-16 Multiple mechanisms may contribute to activation of von Willebrand factor (vWF) adhesiveness by elongational flow at sites of hemostasis, including enhancement of A1 domain exposure within vWF concatamers and conformational change within the A1 domain or its complex with GPIbα. A receptor and ligand in a single molecule (ReaLiSM) containing the A1 domain, a flexible linker, and GPIbα fused in a single polypeptide and suspended between beads using DNA handles was interrogated with a laser trap. Two pathways for unbinding representing flexed and extended states were previously reported, with the flexed, more stable state predominantly at forces above 10 pN. vWD type 2B mutations in the A1 domain selectively stabilize the extended, high affinity state, whereas platelet-type vWD mutations in GPIbα stabilize both states. With ReaLiSM, we can also measure the kinetics and force-dependence of receptor-ligand binding. Remarkably, we also see two on-rates for receptor-ligand binding, with the faster on-rate predominating above 10 pN and the slower on-rate predominating below 10 pN. vWD type 2B mutations in the A1 domain selectively increased the fast on-rate, whereas platelet-type mutations in GPIbα increased both on-rates. Our results support force-dependent conformational change as one of the mechanisms that activates vWF in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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