Watching conformational dynamics of ABC transporters with single-molecule tools

2015 ◽  
Vol 43 (5) ◽  
pp. 1041-1047 ◽  
Author(s):  
Florence Husada ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Gea K. Schuurman-Wolters ◽  
Evelyn Ploetz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Abc Transporters ◽  
Fluorescent Dyes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Cell Volume Regulation ◽  
Size Exclusion ◽  
Conformational States ◽  
Functional Interpretation ◽  
Transport Cycle

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

scholarly journals Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽  
2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Evelyn Ploetz ◽  
Gea K. Schuurman-Wolters ◽  
Niels Zijlstra ◽  
Amarins W. Jager ◽  
Douglas A. Griffith ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
Titration Calorimetry ◽  
Uptake System ◽  
Conformational States ◽  
Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

scholarly journals Single-molecule spectroscopy reveals dynamic allostery mediated by the substrate-binding domain of a AAA+ machine

2020 ◽  
Author(s):  
Marija Iljina ◽  
Hisham Mazal ◽  
Pierre Goloubinoff ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Regulatory Pathways ◽  
Time Dynamics ◽  
Binding Domains ◽  
Substrate Binding Domain

AbstractClpB is an ATP-dependent protein disaggregation machine that is activated on demand by co-chaperones and by aggregates caused by heat shock or mutations. The regulation of ClpB’s function is critical, since its persistent activation is toxic in vivo. Each ClpB molecule is composed of an auxiliary N-terminal domain (NTD), an essential regulatory middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains that are responsible for ATP-fuelled substrate threading. The NTD is generally thought to serve as a substrate-binding domain, which is commonly considered to be dispensable for ClpB’s activity, and is not well-characterized structurally due to its high mobility. Here we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB’s NTD and reveal its involvement in novel allosteric interactions. We find that the NTD fluctuates on a microsecond timescale and, unexpectedly, shows little change in conformational dynamics upon binding of a substrate protein. During its fast motion, the NTD makes crucial contacts with the regulatory MD, directly affecting its conformational state and thereby influencing the overall ATPase and unfolding activity of this machine. Moreover, we also show that the NTD mediates signal transduction to the nucleotide-binding domains through conserved residues. The two regulatory pathways revealed here enable the NTD to suppress the MD in the absence of protein substrate, and to limit ATPase and disaggregation activities of ClpB. The use of multiple parallel allosteric pathways involving ultrafast domain motions might be common to AAA+ molecular machines to ensure their fast and reversible activation.

scholarly journals RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

2021 ◽  
Author(s):  
Abhishek Mazumder ◽  
Anna Wang ◽  
Heesoo Uhm ◽  
Richard H Ebright ◽  
Achillefs N Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Small Molecules ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monovalent Cation ◽  
Structural Element ◽  
Conformational States ◽  
Domains Of Life

Abstract The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

scholarly journals RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

2020 ◽  
Author(s):  
Abhishek Mazumder ◽  
Anna Wang ◽  
Heesoo Uhm ◽  
Richard H. Ebright ◽  
Achillefs N. Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Small Molecules ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monovalent Cation ◽  
Structural Element ◽  
Conformational States ◽  
Domains Of Life

AbstractThe RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

scholarly journals Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Marijn de Boer ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Stephanie L Begg ◽  
Gea K Schuurman-Wolters ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Proteins ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Complex Interplay ◽  
Selective Transport ◽  
Closed Conformation ◽  
Single Molecule Fret ◽  
The Impact ◽  
Dynamic Plasticity

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

scholarly journals Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

2021 ◽  
Author(s):  
Chengcheng Fan ◽  
Douglas C Rees
Keyword(s):  
Abc Transporters ◽  
Ternary Complex ◽  
Conformational States ◽  
Type Iv ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Heavy Metal Detoxification ◽  
Transport Cycle ◽  
Mixed Disulfides ◽  
Glutathione Binding

The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined structures of AtAtm3 in multiple conformational states. These structures not only provide a structural framework for defining the alternating access transport cycle, but also highlight an unappreciated feature of the glutathione binding site, namely the paucity of cysteine residues that could potentially form inhibitory mixed disulfides with glutathione. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 with bound GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures containing both nucleotide and transport substrate suggests that this ternary complex exists only transiently during the transport cycle.

scholarly journals Allosteric Response and Substrate Sensitivity in Peptide Binding of the Signal Recognition Particle

2014 ◽  
Vol 289 (44) ◽  
pp. 30868-30879 ◽  
Author(s):  
Connie Y. Wang ◽  
Thomas F. Miller
Keyword(s):  
Single Molecule ◽  
Protein Targeting ◽  
Substrate Binding ◽  
Body Movement ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Signal Recognition Particle ◽  
Central Component ◽  
Signal Recognition ◽  
Dynamics Simulations

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway.

scholarly journals Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function

2020 ◽  
Vol 117 (14) ◽  
pp. 7814-7823 ◽  
Author(s):  
Si Wu ◽  
Liu Hong ◽  
Yuqing Wang ◽  
Jieqiong Yu ◽  
Jie Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Kinetic Measurements ◽  
Kinetics Of ◽  
Conformational Distribution

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

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