Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of Förster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness.

Download Full-text

Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽

10.3390/molecules23123105 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3105 ◽

Cited By ~ 1

Author(s):

Henning Höfig ◽

Michele Cerminara ◽

Ilona Ritter ◽

Antonie Schöne ◽

Martina Pohl ◽

...

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Fluorescent Dyes ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Strong Increase ◽

Single Molecule Level ◽

Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

Download Full-text

Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

eLife ◽

10.7554/elife.16886 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 24

Author(s):

Ucheor B Choi ◽

Minglei Zhao ◽

Yunxiang Zhang ◽

Ying Lai ◽

Axel T Brunger

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Neurotransmitter Release ◽

Plasma Membranes ◽

Molecular Mechanisms ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Snare Complex ◽

Spontaneous Release ◽

Trans Conformation

Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

Download Full-text

Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation

10.1101/848788 ◽

2019 ◽

Author(s):

Emma Branigan ◽

J. Carlos Penedo ◽

Ronald T. Hay

Keyword(s):

Single Molecule ◽

Active Form ◽

Resonance Energy Transfer ◽

E3 Ligase ◽

Resonance Energy ◽

Post Translational Modification ◽

E3 Ligases ◽

Closed Conformation ◽

Ring E3 Ligase ◽

Ring E3

AbstractUbiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate for catalysis by locking it into a “closed” conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.

Download Full-text

Minimalist Approaches to Protein Labelling: Getting the Most Fluorescent Bang for Your Steric Buck

Australian Journal of Chemistry ◽

10.1071/ch13554 ◽

2014 ◽

Vol 67 (5) ◽

pp. 686 ◽

Cited By ~ 11

Author(s):

Lee C. Speight ◽

Moumita Samanta ◽

E. James Petersson

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Unnatural Amino Acid ◽

Spectroscopic Techniques ◽

Environmental Sensitivity ◽

Protein Labelling ◽

Single Molecule Level ◽

Wavelength Absorption

Fluorescence methods allow one to monitor protein conformational changes, protein–protein associations, and proteolysis in real time, at the single molecule level and in living cells. The information gained in such experiments is a function of the spectroscopic techniques used and the strategic placement of fluorophore labels within the protein structure. There is often a trade-off between size and utility for fluorophores, whereby large size can be disruptive to the protein’s fold or function, but valuable characteristics, such as visible wavelength absorption and emission or brightness, require sizable chromophores. Three major types of fluorophore readouts are commonly used: (1) Förster resonance energy transfer (FRET); (2) photoinduced electron transfer (PET); and (3) environmental sensitivity. This review focuses on those probes small enough to be incorporated into proteins during ribosomal translation, which allows the probes to be placed on the interiors of proteins as they are folded during synthesis. The most broadly useful method for doing so is site-specific unnatural amino acid (UAA) mutagenesis. We discuss the use of UAA probes in applications relying on FRET, PET, and environmental sensitivity. We also briefly review other methods of protein labelling and compare their relative merits to UAA mutagenesis. Finally, we discuss small probes that have thus far been used only in synthetic peptides, but which have unusual value and may be candidates for incorporation using UAA methods.

Download Full-text

Folding and catalysis of the hairpin ribozyme

Biochemical Society Transactions ◽

10.1042/bst0330461 ◽

2005 ◽

Vol 33 (3) ◽

pp. 461-465 ◽

Cited By ~ 24

Author(s):

T.J. Wilson ◽

M. Nahas ◽

T. Ha ◽

D.M.J. Lilley

Keyword(s):

Single Molecule ◽

Ph Dependence ◽

Active Form ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Base Catalysis ◽

Hairpin Ribozyme ◽

Acid Base ◽

Physiological Conditions ◽

Single Molecule Enzymology

The active form of the hairpin ribozyme is brought about by the interaction of two formally unpaired loops. In a natural molecule, these are present on two adjacent arms of a four-way junction. Although activity can be obtained in molecules lacking this junction, the junction is important in the promotion of the folded state of the ribozyme under physiological conditions, at a rate that is faster than the chemical reaction. Single-molecule fluorescence resonance energy transfer studies show that the junction introduces a discrete intermediate into the folding process, which repeatedly juxtaposes the two loops and thus promotes their docking. Using single-molecule enzymology, the cleavage and ligation rates have been measured directly. The pH dependence of the rates is consistent with a role for nucleobases acting in general acid–base catalysis.

Download Full-text

Rhynchosia volubilis Promotes Cell Survival via cAMP-PKA/ERK-CREB Pathway

Pharmaceuticals ◽

10.3390/ph15010073 ◽

2022 ◽

Vol 15 (1) ◽

pp. 73

Author(s):

Sang-Hyun Ahn ◽

Jung-Soo Suh ◽

Yoon-Kwan Jang ◽

Heon-Su Kim ◽

Gyu-Ho Choi ◽

...

Keyword(s):

Cell Survival ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Ethanol Extract ◽

Cellular Effects ◽

Downstream Effectors ◽

Traditional Remedy ◽

Creb Pathway

Rhynchosia volubilis, a small black bean, has been used as a traditional remedy to treat diseases and maintain health in East Asia, but its cellular effects and molecular mechanisms are not fully understood. The purpose of this study was to investigate the effect of ethanol extract from Rhynchosia volubilis (EERV) on cell survival and to elucidate the biochemical signaling pathways. Our results showed that EERV stimulated the cyclic AMP (cAMP) signal revealed by a fluorescent protein (FP)-based intensiometric sensor. Using a Förster resonance energy transfer (FRET)-based sensor, we further revealed that EERV could activate PKA and ERK signals, which are downstream effectors of cAMP. In addition, we reported that EERV could induce the phosphorylation of CREB, a key signal for cell survival. Thus, our results suggested that EERV protects against apoptosis by activating the cell survival pathway through the cAMP-PKA/ERK-CREB pathway.

Download Full-text

Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Download Full-text

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

10.31237/osf.io/9j8gq ◽

2018 ◽

Author(s):

Alexander Carl DeHaven

Keyword(s):

Energy Transfer ◽

Structural Dynamics ◽

Single Molecule ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Förster Resonance Energy Transfer ◽

U2 Snrna ◽

Folding Dynamics ◽

The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

Download Full-text