Name: utilizing time-resolved protein-induced fluorescence enhancement to identify stable local conformations one α-synuclein monomer at a time
Uploaded: 2021-05-30T14:00:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One Î±-Synuclein Monomer at a Time at JoVE.com

The pT-t7 plasmid encoding A56C &#945;-Syn mutant was given to us as a present from Dr. Asaf Grupi, Dr. Dan Amir and Dr. Elisha Haas. This paper was supported by the National Institutes of Health (NIH, grant R01 GM130942 to E.L. as a subaward), the Israel Science Foundation (grant 3565/20 within the KillCorona - Curbing Coronavirus Research Program), the Milner Fund and the Hebrew University of Jerusalem (startup funds).

All authors share no conflict of interest.

Extensive biochemical and biophysical studies were performed to study the structural characteristics of &#945;-Syn and its disordered nature33,34,35,36,37,38. Several works have already utilized freely-diffusing smFRET to investigate the intra-molecular dynamics of the &#945;-Syn monomer free of binding. These works reported ...

Over the past two decades, single-molecule fluorescence-based methods have become a powerful tool for measuring biomolecules1,2, probing how different biomolecular parameters distribute as well as how they dynamically interconvert between different sub-populations of these parameters at sub-millisecond resolution3,4,5. The parameters in these techniques include the energy transfer efficiency in FRET&#160;measurements 6,7, fluorescence anisotropy8,9, fluorescence quantum yields and lifetimes10,11, as a function of different fluorescence quenching12 or enhancement13 mechanisms. One of these mechanisms, better known as protein-induced fluorescence enhancement (PIFE)14 introduces the enhancement of fluorescence quantum yield and lifetime as a function of steric obstruction to the free isomerization of the fluorophore when in excited-state, caused by protein surfaces in the vicinity of the dye14,15,16,17,18,19. Both FRET and PIFE are considered spectroscopic rulers or proximity sensors since their measured parameter is directly linked to a spatial measure within the labeled biomolecule under measurement. While the FRET efficiency is related to the distance between a pair of dyes within a range of 3-10 nm20, PIFE tracks increases in fluorescence quantum yields or lifetimes related to the distance between the dye and a surface of a nearby protein in the range of 0-3 nm19.
Single-molecule FRET has been widely used for providing structural insights into many different protein systems, including intrinsically disordered proteins (IDPs)21, such as &#945;-Synuclein (&#945;-Syn)22. &#945;-Syn can form ordered structures following binding to different biomolecules and under different conditions23,24,25,26,27,28,29,30. However, when unbound, the &#945;-Syn monomer is characterized by high conformational heterogeneity with rapidly interconverting conformations31,32.
The conformations of &#945;-Syn have been studied previously using various different techniques that help in identifying conformational dynamics of such highly heterogeneous and dynamic protein systems33,34,35,36,37,38,39. Interestingly, single-molecule FRET (smFRET) measurements of &#945;-Syn in buffer reported a single FRET population39,40&#160;that is an outcome of time-averaging of conformations dynamically interconverting at times much faster than the typical diffusion time of &#945;-Syn through the confocal spot (times as fast as few microseconds and even faster than that, relative to typical millisecond diffusion times)40,41. However, using a FRET spectroscopic ruler with the 3-10 nm distance sensitivity sometimes reports only on overall structural changes in a small protein such as &#945;-Syn. Single-molecule measurements utilizing spectroscopic proximity sensors with shorter distance sensitivities have the potential to report on dynamics of local structures. Herein we perform single-molecule PIFE measurements of &#945;-Syn and identify different sub-populations of fluorescence lifetimes mapping to different local structures with transitions between them as slow as 100 ms. This work summarizes time-resolved smPIFE measurements of freely-diffusing &#945;-Syn molecules one at a time, in buffer and when bound to SDS-based membranes as a short-range single-molecule spectroscopic proximity sensor.

<table><tbody><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Merc</td><td>C7715</td><td>cutoff: 100 kDa</td></tr><tr><td>ammonium sulfate</td><td>Sigma-Aldrich</td><td>A4418</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>cysteamine</td><td>Sigma-Aldrich</td><td>30070</td><td></td></tr><tr><td>dialysis bags - MEGA GeBaFlex-tube</td><td>Gene Bio-Application</td><td>MEGA320</td><td></td></tr><tr><td>dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Fast SeeBand staining solution</td><td>Gene Bio-Application</td><td>SB050</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>50046</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>54457</td><td></td></tr><tr><td>HiTrap Desalting 5 mL</td><td>Sigma-Aldrich</td><td>GE17-1408</td><td></td></tr><tr><td>6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX)</td><td>Sigma-Aldrich</td><td>238813</td><td></td></tr><tr><td>isopropyl &amp;beta;-d-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>LB broth</td><td>Sigma-Aldrich</td><td>L3152</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>63068</td><td></td></tr><tr><td>MonoQ column</td><td>Sigma-Aldrich</td><td>54807</td><td></td></tr><tr><td>protein LoBind tube</td><td>Sigma-Aldrich</td><td>EP0030108094</td><td>0.5 mL</td></tr><tr><td>Rinse a &amp;micro;-slide 18</td><td>Ibidi</td><td>81816</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>75746</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma-Aldrich</td><td>71507</td><td></td></tr><tr><td>Sterile Cell spreaders, Drigalski spatulas</td><td>mini-plast</td><td>815-004-05-001</td><td></td></tr><tr><td>streptomycin sulfate</td><td>Sigma-Aldrich</td><td>S9137</td><td></td></tr><tr><td>sulfo-Cy3&amp;nbsp;maleimide</td><td>abcam</td><td>ab146493</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Sigma-Aldrich</td><td>75259</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>93363</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293</td><td></td></tr><tr><td>Yeast Extract</td><td>Sigma-Aldrich</td><td>Y1625</td><td></td></tr></tbody></table>

utilizing time-resolved protein-induced fluorescence enhancement to identify stable local conformations one &#945;-synuclein monomer at a time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

As an IDP, when it is not bound to another biomolecule, &#945;-Syn exhibits structural dynamics between multiple conformations, with transitions at few microseconds40&#160;and even at hundreds of nanoseconds41. When &#945;-Syn crosses the confocal spot, it may undergo thousands of transitions between conformations. Indeed, this was the case when smFRET was used39,40. Here we perform smPIFE measurements in order to p...

Watch this Scientific Journal Video about Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One Î±-Synuclein Monomer at a Time at JoVE.com

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

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Research

JoVE Journal

Biochemistry

3.1K Views.  The Hebrew University of Jerusalem. Single-molecule, eiwit-geïnduceerde fluorescentieverbetering kan een aanvulling zijn op single-molecule FRET-metingen bij het bestuderen van eiwitstructure subpopulaties en conformaties, vooral in gevallen waarin verschillende structurele subpopulaties rapporteren over stabiele lokale structuren. Het belangrijkste voordeel van deze techniek is dat het verschillende site-specifieke structurele subpopulaties vangt op basis van de nabijheid van de kleurstofetiketteringsplaats naar de eiwitopper...

Video: Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Monitoring GTPase Atlastin in Different Nucleotide Loading States

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the local flexibility and dynamics of the deoxyribonucleic acid (DNA)-binding forkhead (FKH) domain of the FoxP1 transcription factor. FoxP1 dimerizes through a three-dimensional domain-swapping (3D-DS) mechanism, forming a disordered intermediate with or without DNA. Since 3D-DS involves an intrinsically disordered region, understanding its behavior is crucial for elucidating the structural and functional properties of FoxP1. Using a single-cysteine-labeled FoxP1, we conducted single-molecule fluorescence anisotropy (smFA) experiments, applying dynamic anisotropy Photon Distribution Analysis (daPDA) and time-resolved anisotropy Burst Variance Analysis (traBVA) approaches to probe local flexibility and dynamics. This protocol provides a detailed, step-by-step guide for smFA measurements, emphasizing time-resolved analyses, variance, and probability distribution techniques to capture structural dynamics across different timescales. This approach enabled us to relate dynamics and heterogeneity to FoxP1 dimerization and DNA binding, highlighting the complex action mechanism that characterizes this transcription factor.

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Neuroscience

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

Biology

Live Cell Single-Molecule Imaging-Based Quantification of Protein Interactions

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

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