Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events

Gunnarsson, Anders; Dexlin, Linda; Wallin, Patric; Svedhem, Sofia; Jönsson, Peter; Wingren, Christer; Höök, Fredrik

doi:10.1021/ja2047039

Cited by 27 publications

(32 citation statements)

References 37 publications

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“…As previously described, we are able to determine kinetic rate constants by following the attachment and detachment of labeled liposomes, i.e. analyzing equilibrium fluctuations of single liposome binding events273335. In short, counting newly arrived liposomes over time gives the association rate constant, whilst analyzing the residence time of bound vesicles reveals the dissociation rate constant.…”

Section: Resultsmentioning

confidence: 99%

“…In short, counting newly arrived liposomes over time gives the association rate constant, whilst analyzing the residence time of bound vesicles reveals the dissociation rate constant. (For details see Methods and Gunnarsson et al333435.) A key advantage of this approach is that we are able to study kinetics under steady-state conditions, thus avoiding diffusion limitations.…”

Section: Resultsmentioning

confidence: 99%

“…Images were processed and analyzed with a MatLab (MathWorks, Inc., Natick, Massachusetts, USA) software package as described in detail elsewhere333435. Briefly, a liposome was registered if its intensity exceeded a preset threshold, and was considered as bound if it was present for > 1.2 sec.…”

Section: Methodsmentioning

confidence: 99%

“…Other techniques like atomic force microscopy (AFM) or micropipette aspiration technique provide the required sensitivity, but can only be applied under transient conditions. We recently demonstrated an approach based on total internal reflection fluorescence (TIRF) microscopy as a versatile tool for the study of single liposome binding events allowing for the detection of association and dissociation events under equilibrium binding conditions27333435.…”

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confidence: 99%

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Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions

Kunze

Bally

Höök

et al. 2013

Sci Rep

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Carbohydrate−carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Lex glycosphingolipids (Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), which were employed to mimic cell−cell contacts. The kinetic parameters of the self-interaction between Lex-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions

Kunze

Bally

Höök

et al. 2013

Sci Rep

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“…Binding to the functionalized surfaces was recorded using total internal reflection fluorescence (TIRF) microscopy. From the recorded movies, we extracted information on HSV mobility using single-particle tracking (SPT), and on binding kinetics using equilibrium fluctuation analysis (30,31).…”

Section: Introductionmentioning

confidence: 99%

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans

Peerboom

Block

Altgärde

et al. 2017

Biophysical Journal

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Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

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Identifying Multiple Populations from Single‐Molecule Lifetime Distributions

Kastantin

Schwartz

2012

ChemPhysChem

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A major advantage of single-molecule methods over ensemble-averaging techniques involves the ability to characterize heterogeneity through the identification of multiple molecular populations. It can be challenging, however, to determine absolute values of dynamic parameters (and to relate these values to those determined from a conventional method) because characteristic timescales of various populations may vary over many orders of magnitude, and under a given set of experimental conditions instrumental sensitivity to various populations may be unequal. Using data obtained from the single-molecule tracking microscopy of fibrinogen protein adsorption and desorption, it is shown that by performing a combined analysis of molecular trajectories obtained using a range of acquisition times, it is possible to extract quantitative absolute values of multiple population fractions and residence times (with well-defined uncertainties), even when these values span many orders of magnitude. In particular, as many as six distinct populations are rigorously identified, exhibiting characteristic timescales that vary over nearly three orders of magnitude with population fractions as small as one part in a thousand. This approach will lead to better comparability between single-molecule experiments and may be useful in connecting single-molecule to ensemble-averaged observations.

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Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events

Cited by 27 publications

References 37 publications

Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions

Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans

Identifying Multiple Populations from Single‐Molecule Lifetime Distributions

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