@article{doi:10.1021/acschemneuro.4c00185,

title = {Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils},

author = {Derrick Lau and Yuan Tang and Vijaya Kenche and Thomas Copie and Daryan Kempe and Eve Jary and Noah J. Graves and Maté Biro and Colin L. Masters and Nicolas Dzamko and Yann Gambin and Emma Sierecki},

url = {https://doi.org/10.1021/acschemneuro.4c00185},

doi = {10.1021/acschemneuro.4c00185},

year  = {2024},

date = {2024-08-24},

journal = {ACS Chemical Neuroscience},

volume = {0},

number = {0},

pages = {null},

note = {PMID: 39197832},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Houx2024.05.05.592604,

title = {Quantification of nanocondensates formation at the single molecule level},

author = {Justin Houx and Thomas Copie and Yann Gambin and Emma Sierecki},

url = {https://www.biorxiv.org/content/early/2024/05/06/2024.05.05.592604},

doi = {10.1101/2024.05.05.592604},

year  = {2024},

date = {2024-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy. We demonstrate how straightforward fingerprinting of individual nanocondensates provides an exquisite quantification of their formation, size, density, and their temporal evolution. Overall, this study highlights the potential of single molecule spectroscopy to investigate the formation of biomolecular condensates and liquid-liquid phase separation mechanisms in protein systems.Competing Interest StatementThe authors have declared no competing interest.TDP-43TAR DNA-binding protein 43TARTrans-activation response elementLLPSLiquid-liquid phase separationLCDlow complexity domainTMAOTrimethylamine N-oxideLTELeishmania tarentolae extractTLTop leftTRTop rightBLBottom leftBRBottom rightFUSFused in sarcomacGAScyclic GMP–AMP synthase},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ijms241512134,

title = {α-Synuclein Strains and Their Relevance to Parkinson’s Disease, Multiple System Atrophy, and Dementia with Lewy Bodies},

author = {Noah J. Graves and Yann Gambin and Emma Sierecki},

url = {https://www.mdpi.com/1422-0067/24/15/12134},

doi = {10.3390/ijms241512134},

issn = {1422-0067},

year  = {2023},

date = {2023-01-01},

journal = {International Journal of Molecular Sciences},

volume = {24},

number = {15},

abstract = {Like many neurodegenerative diseases, Parkinson’s disease (PD) is characterized by the formation of proteinaceous aggregates in brain cells. In PD, those proteinaceous aggregates are formed by the α-synuclein (αSyn) and are considered the trademark of this neurodegenerative disease. In addition to PD, αSyn pathological aggregation is also detected in atypical Parkinsonism, including Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), as well as neurodegeneration with brain iron accumulation, some cases of traumatic brain injuries, and variants of Alzheimer’s disease. Collectively, these (and other) disorders are referred to as synucleinopathies, highlighting the relation between disease type and protein misfolding/aggregation. Despite these pathological relationships, however, synucleinopathies cover a wide range of pathologies, present with a multiplicity of symptoms, and arise from dysfunctions in different neuroanatomical regions and cell populations. Strikingly, αSyn deposition occurs in different types of cells, with oligodendrocytes being mainly affected in MSA, while aggregates are found in neurons in PD. If multiple factors contribute to the development of a pathology, especially in the cases of slow-developing neurodegenerative disorders, the common presence of αSyn aggregation, as both a marker and potential driver of disease, is puzzling. In this review, we will focus on comparing PD, DLB, and MSA, from symptomatology to molecular description, highlighting the role and contribution of αSyn aggregates in each disorder. We will particularly present recent evidence for the involvement of conformational strains of αSyn aggregates and discuss the reciprocal relationship between αSyn strains and the cellular milieu. Moreover, we will highlight the need for effective methodologies for the strainotyping of aggregates to ameliorate diagnosing capabilities and therapeutic treatments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1021/acschemneuro.1c00553,

title = {Single Molecule Fingerprinting Reveals Different Amplification Properties of α-Synuclein Oligomers and Preformed Fibrils in Seeding Assay},

author = {Derrick Lau and Chloé Magnan and Kathryn Hill and Antony Cooper and Yann Gambin and Emma Sierecki},

url = {https://doi.org/10.1021/acschemneuro.1c00553},

doi = {10.1021/acschemneuro.1c00553},

year  = {2022},

date = {2022-01-01},

journal = {ACS Chemical Neuroscience},

volume = {13},

number = {7},

pages = {883-896},

note = {PMID: 35286811},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Leitão2021,

title = {Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein},

author = {André D. G. Leitão and Paulina Rudolffi-Soto and Alexandre Chappard and Akshay Bhumkar and Derrick Lau and Dominic J. B. Hunter and Yann Gambin and Emma Sierecki},

doi = {10.1038/s42003-021-02624-x},

issn = {2399-3642},

year  = {2021},

date = {2021-12-00},

journal = {Commun Biol},

volume = {4},

number = {1},

publisher = {Springer Science and Business Media LLC},

abstract = {AbstractThe aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Brown2019,

title = {Single-molecule detection on a portable 3D-printed microscope},

author = {James W. P. Brown and Arnaud Bauer and Mark E Polinkovsky and Akshay Bhumkar and Dominic J. B. Hunter and Katharina Gaus and Emma Sierecki and Yann Gambin},

doi = {10.1038/s41467-019-13617-0},

issn = {2041-1723},

year  = {2019},

date = {2019-12-00},

journal = {Nat Commun},

volume = {10},

number = {1},

publisher = {Springer Science and Business Media LLC},

abstract = {AbstractSingle-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{O’Carroll2018,

title = {Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88},

author = {Ailís O’Carroll and Brieuc Chauvin and James W. P. Brown and Ava Meagher and Joanne Coyle and Jurgen Schill and Akshay Bhumkhar and Dominic J. B. Hunter and Thomas Ve and Bostjan Kobe and Emma Sierecki and Yann Gambin},

doi = {10.1186/s12915-018-0611-7},

issn = {1741-7007},

year  = {2018},

date = {2018-12-00},

journal = {BMC Biol},

volume = {16},

number = {1},

publisher = {Springer Science and Business Media LLC},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Moustaqil2018,

title = {Homodimerization regulates an endothelial specific signature of the SOX18 transcription factor},

author = {Mehdi Moustaqil and Frank Fontaine and Jeroen Overman and Alex McCann and Timothy L Bailey and Paulina Rudolffi Soto and Akshay Bhumkar and Nichole Giles and Dominic J B Hunter and Yann Gambin and Mathias Francois and Emma Sierecki},

doi = {10.1093/nar/gky897},

issn = {1362-4962},

year  = {2018},

date = {2018-11-30},

volume = {46},

number = {21},

pages = {11381--11395},

publisher = {Oxford University Press (OUP)},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Jung2018,

title = {Cell-free formation and interactome analysis of caveolae},

author = {WooRam Jung and Emma Sierecki and Michele Bastiani and Ailis O’Carroll and Kirill Alexandrov and James Rae and Wayne Johnston and Dominic J.B. Hunter and Charles Ferguson and Yann Gambin and Nicholas Ariotti and Robert G. Parton},

doi = {10.1083/jcb.201707004},

issn = {1540-8140},

year  = {2018},

date = {2018-06-04},

volume = {217},

number = {6},

pages = {2141--2165},

publisher = {Rockefeller University Press},

abstract = {Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)–stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2018,

title = {Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC},

author = {Yann Gambin and Nichole Giles and Ailís O'Carroll and Mark Polinkovsky and Dominic Hunter and Emma Sierecki},

doi = {10.1016/j.jmb.2017.12.013},

issn = {0022-2836},

year  = {2018},

date = {2018-02-00},

journal = {Journal of Molecular Biology},

volume = {430},

number = {4},

pages = {491--508},

publisher = {Elsevier BV},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2018b,

title = {Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC},

author = {Yann Gambin and Nichole Giles and Ailís O'Carroll and Mark Polinkovsky and Dominic Hunter and Emma Sierecki},

doi = {10.1016/j.jmb.2017.12.013},

issn = {0022-2836},

year  = {2018},

date = {2018-02-00},

journal = {Journal of Molecular Biology},

volume = {430},

number = {4},

pages = {491--508},

publisher = {Elsevier BV},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sierecki2016,

title = {Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence},

author = {Emma Sierecki and Nichole Giles and Quill Bowden and Mark E. Polinkovsky and Janina Steinbeck and Nicholas Arrioti and Diya Rahman and Akshay Bhumkar and Philip R. Nicovich and Ian Ross and Robert G. Parton and Till Böcking and Yann Gambin},

doi = {10.1038/srep37630},

issn = {2045-2322},

year  = {2016},

date = {2016-12-23},

journal = {Sci Rep},

volume = {6},

number = {1},

publisher = {Springer Science and Business Media LLC},

abstract = {AbstractProtein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2016b,

title = {Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide},

author = {Yann Gambin and Mark Polinkovsky and Bill Francois and Nichole Giles and Akshay Bhumkar and Emma Sierecki},

doi = {10.3390/ijms17050655},

issn = {1422-0067},

year  = {2016},

date = {2016-05-00},

journal = {IJMS},

volume = {17},

number = {5},

publisher = {MDPI AG},

abstract = {Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2016,

title = {Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide},

author = {Yann Gambin and Mark Polinkovsky and Bill Francois and Nichole Giles and Akshay Bhumkar and Emma Sierecki},

doi = {10.3390/ijms17050655},

issn = {1422-0067},

year  = {2016},

date = {2016-05-00},

journal = {IJMS},

volume = {17},

number = {5},

publisher = {MDPI AG},

abstract = {Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sierecki2014,

title = {Rapid Mapping of Interactions between Human SNX-BAR Proteins Measured In Vitro by AlphaScreen and Single-molecule Spectroscopy},

author = {Emma Sierecki and Loes M. Stevers and Nichole Giles and Mark E. Polinkovsky and Mehdi Moustaqil and Sergey Mureev and Wayne A. Johnston and Mareike Dahmer-Heath and Dubravka Skalamera and Thomas J. Gonda and Brian Gabrielli and Brett M. Collins and Kirill Alexandrov and Yann Gambin},

doi = {10.1074/mcp.m113.037275},

issn = {1535-9476},

year  = {2014},

date = {2014-09-00},

journal = {Molecular & Cellular Proteomics},

volume = {13},

number = {9},

pages = {2233--2245},

publisher = {Elsevier BV},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1126/science.1249783,

title = {Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor},

author = {Andrew J. Brooks and Wei Dai and Megan L. O’Mara and Daniel Abankwa and Yash Chhabra and Rebecca A. Pelekanos and Olivier Gardon and Kathryn A. Tunny and Kristopher M. Blucher and Craig J. Morton and Michael W. Parker and Emma Sierecki and Yann Gambin and Guillermo A. Gomez and Kirill Alexandrov and Ian A. Wilson and Manolis Doxastakis and Alan E. Mark and Michael J. Waters},

url = {https://www.science.org/doi/abs/10.1126/science.1249783},

doi = {10.1126/science.1249783},

year  = {2014},

date = {2014-01-01},

journal = {Science},

volume = {344},

number = {6185},

pages = {1249783},

abstract = {Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors. A molecular mechanism for transmembrane signaling by the growth hormone receptor is elucidated. [Also see Perspective by Wells and Kossiakoff] The receptor for growth hormone is a well-studied representative of a family of cytokine receptors through which binding of hormone molecules at the cell surface is converted into a biochemical signal within the cell. Brooks et al. (10.1126/science.1249783; see the Perspective by Wells and Kossiakoff) used a combination of crystal structures, biophysical measurements, cell biology experiments with modified receptors, and molecular dynamics and modeling to decipher how the receptor actually transmits the information that a hormone molecule is bound. The results suggest that the receptors exist in inactive dimeric complexes in which two associated JAK2 protein kinase molecules interact in an inhibitory manner. Binding of growth hormone causes a structural change in the receptor that results in movement of the receptor intracellular domains apart from one another. This relieves the inhibition of the JAK2 molecules and allows them to activate one another, thus initiating the cellular response to the hormone.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.7554/eLife.01434,

title = {Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae},

author = {Yann Gambin and Nicholas Ariotti and Kerrie-Ann McMahon and Michele Bastiani and Emma Sierecki and Oleksiy Kovtun and Mark E Polinkovsky and Astrid Magenau and WooRam Jung and Satomi Okano and Yong Zhou and Natalya Leneva and Sergey Mureev and Wayne Johnston and Katharina Gaus and John F Hancock and Brett M Collins and Kirill Alexandrov and Robert G Parton},

editor = {Suzanne R Pfeffer},

url = {https://doi.org/10.7554/eLife.01434},

doi = {10.7554/eLife.01434},

issn = {2050-084X},

year  = {2014},

date = {2014-01-01},

journal = {eLife},

volume = {3},

pages = {e01434},

publisher = {eLife Sciences Publications, Ltd},

abstract = {In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ferreon2012,

title = {Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape},

author = {Allan Chris M. Ferreon and Mahdi Muhammad Moosa and Yann Gambin and Ashok A. Deniz},

doi = {10.1073/pnas.1201802109},

issn = {1091-6490},

year  = {2012},

date = {2012-10-30},

journal = {Proc. Natl. Acad. Sci. U.S.A.},

volume = {109},

number = {44},

pages = {17826--17831},

publisher = {Proceedings of the National Academy of Sciences},

abstract = {Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson’s disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 2∶1 [urea]∶[TMAO] ratio has a net neutral effect on the protein’s dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2010,

title = {Ultrafast microfluidic mixer with three-dimensional flow focusing for studies of biochemical kinetics},

author = {Yann Gambin and Claire Simonnet and Virginia VanDelinder and Ashok Deniz and Alex Groisman},

doi = {10.1039/b914174j},

issn = {1473-0189},

year  = {2010},

date = {2010-00-00},

journal = {Lab Chip},

volume = {10},

number = {5},

pages = {598--609},

publisher = {Royal Society of Chemistry (RSC)},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gambin2009,

title = {Direct single-molecule observation of a protein living in two opposed native structures},

author = {Yann Gambin and Alexander Schug and Edward A. Lemke and Jason J. Lavinder and Allan Chris M. Ferreon and Thomas J. Magliery and José N. Onuchic and Ashok A. Deniz},

doi = {10.1073/pnas.0904461106},

issn = {1091-6490},

year  = {2009},

date = {2009-06-23},

journal = {Proc. Natl. Acad. Sci. U.S.A.},

volume = {106},

number = {25},

pages = {10153--10158},

publisher = {Proceedings of the National Academy of Sciences},

abstract = {Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (smFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1073/pnas.0809232106,

title = {Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence},

author = {Allan Chris M. Ferreon and Yann Gambin and Edward A. Lemke and Ashok A. Deniz},

url = {https://www.pnas.org/doi/abs/10.1073/pnas.0809232106},

doi = {10.1073/pnas.0809232106},

year  = {2009},

date = {2009-01-01},

journal = {Proceedings of the National Academy of Sciences},

volume = {106},

number = {14},

pages = {5645-5650},

abstract = {We studied the coupled binding and folding of α-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of α-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple α-helical structures. α-Synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive α-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the α-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of α-synuclein.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}