Single molecule fluorescence for membrane proteins

Castell, Oliver K.; Dijkman, Patricia M.; Wiseman, Daniel N.; Goddard, Alan D.

doi:10.1016/j.ymeth.2018.05.024

Cited by 16 publications

(17 citation statements)

References 69 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, there is a growing need for developing new approaches to investigate membrane proteins dynamics (Konermann et al, 2011;Smith et al, 2012;Sim et al, 2017;Mandala et al, 2018;Burke, 2019). These include a combination of molecular dynamics (MD) simulations (Roux et al, 2011;Zdravkovic et al, 2012;Zhekova et al, 2016;Zhekova et al, 2017;Harpole and Delemotte, 2018) with advanced experimental techniques, including spectroscopic techniques and single-particle cryogenic electron microscopy (Smith et al, 2012;Pandey et al, 2016;Castell et al, 2018;Hagn et al, 2018;Ravula and Ramamoorthy, 2019;Sun and Gennis, 2019).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

Giladi

Khananshvili

2020

Front. Pharmacol.

View full text Add to dashboard Cite

Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and highresolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange massspectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding. FIGURE 1 | Schematic overview of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) workflow. Proteins are labeled in D 2 O buffer for a predefined time, followed by exchange quenching and proteolysis. The peptides are separated, and their mass is detected using MS. Finally, the amount of incorporated deuterium within each peptide is calculated for each time point. Giladi and Khananshvili HDX-MS of Membrane Transporters Frontiers in Pharmacology | www.frontiersin.org

show abstract

Section: Introductionmentioning

confidence: 99%

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

Giladi

Khananshvili

2020

Front. Pharmacol.

View full text Add to dashboard Cite

show abstract

“…The spatially confined excitation and photobleaching in TIRF (TIRF‐PB) has been instrumental to achieve an axial superresolution in densely stained probes , or for counting subunits of proteins in single molecular complexes . In either case, the excitation intensity and absence of imaging artifacts are critical to avoid motion artifacts and to apply secondary image processing algorithms.…”

Section: Analysis Of Residence Times Of Membrane‐associated Proteinsmentioning

confidence: 99%

“…TIRF illumination through objectives with high numerical aperture, commonly referred to as objective‐type TIRF microscopy, has become a widespread standard to image signaling events during stimulation of cells with neurotransmitters, hormones, or other modulators . Owing to the efficient suppression of background signals, TIRF microscopy has become a frequently applied method for single molecule detection, tracking and stepwise photobleaching to directly count subunits in oligomeric protein assemblies . The potential to achieve an axial superresolution has been further exploited by varying the AOI with or without sequential photobleaching .…”

Section: Introductionmentioning

confidence: 99%

“…The potential to achieve an axial superresolution has been further exploited by varying the AOI with or without sequential photobleaching . Finally TIRF microscopy can be combined with other methods to detect molecular proximity by Förster resonance energy transfer , to follow lateral mobility by correlation spectroscopy or fluorescence redistribution after photobleaching , to assess the orientation of fluorochromes by polarized TIRF microscopy, to enhance the signal noise ratio by multiphoton excitation , or to achieve lateral superresolution by means of structured TIRF illumination . Since some of these techniques critically rely on mathematical processing, artifact‐free imaging with best possible signal‐to‐noise ratio is highly desirable.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

Schaefer

2020

Journal of Biophotonics

View full text Add to dashboard Cite

The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective-type TIRF microscopy can be achieved by circular or multi-spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light-emitting diode (LED)-based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED-TIRF remain the same as in laser-based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED-TIRF. Using state-of-the art high power LED emitters, LED-TIRF achieves excitation efficiencies that are comparable to laser-based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching-assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane-attached fusion proteins, we provide examples for a photobleaching-based assessment of the exchange kinetics of proteins within living human endothelial cells.

show abstract

“…These issues plague the study of membrane protein outside the membrane. Fluorescence labeling is commonly used, but it is not feasible for small molecules, which poses a problem, particularly to pharmacology, since nearly 90% of drugs are small molecules [ 5 ]. Radiolabeling works without affecting binding kinetics, even with small molecules, but it is extremely costly and involves the hazards of radioactive materials.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Quantification of Protein Binding Kinetics in Whole Cells with Surface Plasmon Resonance Imaging and Edge Deformation Tracking

et al. 2020

View full text Add to dashboard Cite

Most drugs work by binding to receptors on the cell surface. Quantification of binding kinetics between drug and membrane protein is an essential step in drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, and both have issues. Surface plasmon resonance (SPR) imaging has been demonstrated for quantification of protein binding to cells with single-cell resolution, but it only senses the bottom of the cell and the signal diminishes with the molecule size. We have discovered that ligand binding to the cell surface is accompanied by a small cell membrane deformation, which can be used to measure the binding kinetics by tracking the cell edge deformation. Here, we report the first integration of SPR imaging and cell edge tracking methods in a single device, and we use lectin interaction as a model system to demonstrate the capability of the device. The integration enables the simultaneous collection of complementary information provided by both methods. Edge tracking provides the advantage of small molecule binding detection capability, while the SPR signal scales with the ligand mass and can quantify membrane protein density. The kinetic constants from the two methods were cross-validated and found to be in agreement at the single-cell level. The variation of observed rate constant between the two methods is about 0.009 s−1, which is about the same level as the cell-to-cell variations. This result confirms that both methods can be used to measure whole-cell binding kinetics, and the integration improves the reliability and capability of the measurement.

show abstract

Single molecule fluorescence for membrane proteins

Cited by 16 publications

References 69 publications

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

Simultaneous Quantification of Protein Binding Kinetics in Whole Cells with Surface Plasmon Resonance Imaging and Edge Deformation Tracking

Contact Info

Product

Resources

About