This work was supported by the National Natural Science Foundation of China [Grant 32071227 to Z.Y.], Tianjin Municipal Natural Science Foundation of China (22JCYBJC01070 to Z.Y.), and State Key Laboratory of Precision Measuring Technology and Instruments (Tianjin University) [Grant pilab2210 to Z.Y.].

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

The authors declare no competing financial interests or other conflicts of interest.

This protocol employs magnetic tweezers for the manipulation of TRFs at the single-molecule level57,58,59. We utilize magnetic beads to separate TRFs from genomic DNA fragments. Following restriction digestion, TRFs bind to the magnetic beads, enabling their easy separation from genomic DNA fragments. This approach allows for manipulation using magnetic tweezers, which can effectively trap magnetic beads, unlike optical tweezers...

Telomeres are protective structures at the ends of chromosomes1,2,3. Telomere erosion during cell division leads to cell senescence and aging, while abnormal elongation of telomeres contributes to cancer4,5. For telomeres to function properly, their replication, elongation, and damage responses must be highly regulated6,7,8. Shelterin, composed of six subunits, plays a central role in telomere protection9,10,11. A deeper understanding of telomeres will provide valuable insights into telomere biology.
TRF1 and TRF2, core subunits of shelterin, are telomeric binding proteins12,13. Both TRF1 and TRF2 bind to the repetitive DNA motif TTAGGG in telomeres via their Myb domains14. They form dimers through their shared TRFH domains, which allow them to encircle telomeric double-stranded DNA and to recruit telomeric proteins15,16,17,18,19. TRF2 is particularly important for the formation of telomeric D-loops and T-loops20,21. Due to their crucial roles in telomere protection, TRF1 and TRF2 have emerged as potential anti-cancer drug targets22,23,24,25.
Significant efforts have been made to investigate the protein-DNA interactions at telomeres. Biochemical methods such as Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) have been used to examine binding affinities20,26. Numerous structures of telomeric binding proteins complexed with DNA have been elucidated using cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR)27,28,29. Super-resolution imaging techniques like stochastic optical reconstruction microscopy (STORM) have revealed TRF2-dependent T-loop formation21. Recently, nanopore sequencing has been developed to profile telomeric sequences4,30,31. These structural insights have greatly enhanced our understanding of telomeric protein-DNA interactions. To further explore the dynamics of telomeric protein-DNA interactions, the development of new technologies is essential.
Single-molecule tools are powerful techniques for exploring protein-DNA interactions at telomere32,33,34. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers and AFM, have been employed to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin and observe processive telomerase catalysis, among other applications35,36,37,38,39,40. These methods are particularly useful for probing topological conformations and the kinetics of protein-DNA association and dissociation.
However, the preparation of single-molecule constructs with telomeric repetitive motifs still presents challenges, which limits studies using single-molecule mechanical methods. To address this limitation, we have developed a single-molecule mechanical method to study global protein-DNA interactions on full-length human telomeres41. This method directly extracts telomeric DNA from human cells, circumventing the laborious preparation of artificial telomeric DNA. It facilitates the investigation of kinetic processes on long native telomeres spanning several kilobases.
In this protocol, we provide a detailed description of the steps for probing telomeric protein-DNA interactions using magnetic tweezers, a popular single-molecule mechanical tool42,43,44. We demonstrate how to express and purify telomeric proteins, using TRF2 as an example, and how to prepare telomeric DNA from human cells. Additionally, we show how to set up a single-molecule assay on magnetic tweezers to study telomeric protein-DNA interactions, and we cover the subsequent data analysis of single-molecule experiments. This protocol will benefit researchers in the field of telomere biology and telomere-targeted drug discovery.

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			<td>R0568S</td>
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			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>V900933</td>
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			<td>CMOS camera&#160;</td>
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			<td>R0640S</td>
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			<td>Jena Bioscience</td>
			<td>NU-803-DIGXL</td>
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			<td>Dnase I, Rnase-Free, Hc Ea</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0523</td>
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			<td>dNTP mixture</td>
			<td>Nanjing Vazyme Biotech Co., Ltd (Vazyme)</td>
			<td>P032-02</td>
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			<td>DTT</td>
			<td>Solarbio</td>
			<td>D1070</td>
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			<td>Dynabeads M-270&#160; beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>65305</td>
			<td>Streptavidin beads</td>
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		<tr>
			<td>Dynabeads MyOne beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>65001</td>
			<td>Streptavidin beads</td>
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			<td>Ethanol</td>
			<td>Tianjin No.6 Chemical Reagent Factory</td>
			<td>1083</td>
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			<td>Glycerol</td>
			<td>Beijing Hwrkchemical Co,. Ltd</td>
			<td>SMG66258-1</td>
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			<td>Imidazole</td>
			<td>Solarbio</td>
			<td>II0070</td>
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			<td>IPTG</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
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			<td>Isopropanol</td>
			<td>Tianjin No.6 Chemical Reagent Factory</td>
			<td>A1079</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0523</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow fragment (3&#8242;-5&#8242; exo-)</td>
			<td>New England Biolab (NEB)</td>
			<td>M0212S</td>
			<td></td>
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			<td>LabView</td>
			<td>National Instruments</td>
			<td>https://www.ni.com/en-us/shop/product/labview.html</td>
			<td>Graphical programming software&#160;</td>
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			<td>LiCl</td>
			<td>Bide Pharmatech Co., Ltd (bidepharm)</td>
			<td>BD136449</td>
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			<td>Lysozyme</td>
			<td>Solarbio</td>
			<td>L8120-5</td>
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			<td>MseI</td>
			<td>New England Biolab (NEB)</td>
			<td>R0525S</td>
			<td></td>
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			<td>NaCl</td>
			<td>Shanghai Aladdin</td>
			<td>C111533</td>
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			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Spectrophotometer</td>
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			<td>NdeI</td>
			<td>New England Biolab (NEB)</td>
			<td>R0111S</td>
			<td></td>
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			<td>Ni NTA Beads 6FF</td>
			<td>Changzhou Smart-Lifesciences Biotechnology Co.,Ltd</td>
			<td>SA005025</td>
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			<td>Nitrocellulose membrane</td>
			<td>ABclonal</td>
			<td>RM02801</td>
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			<td>PMSF</td>
			<td>Solarbio</td>
			<td>P8340</td>
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			<td>Proteinase K</td>
			<td>Beyotime Biotech Inc (beyotime)</td>
			<td>ST535-500mg</td>
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			<td>rCutSmart Buffer</td>
			<td>New England Biolab (NEB)</td>
			<td>B6004S</td>
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			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R4875</td>
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			<td>Sodium acetate</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>2124902</td>
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			<td>Sumo protease</td>
			<td>Beyotime Biotech Inc (beyotime)</td>
			<td>P2312M</td>
			<td></td>
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analyzing telomeric protein-dna interactions using single-molecule magnetic tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Figure 1A illustrates the schematic domains and structures of TRF1 and TRF2, consisting of 439 and 542 amino acids, respectively, which can be expressed in prokaryotic cells. The preparation of TRF1 has been previously described in the literature41. Here, we provide a comprehensive description and representative results of the preparation of TRF2. Figure 1B shows the plasmid map used for expressing TRF2 in E. coli. We evaluated T...

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

analyzing-telomeric-protein-dna-interactions-using-single-molecule

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

556 Views.  Nankai University. This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Video: Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...