Multivalent interactions, i.e., multiple, non-covalent bonds acting in parallel, are typical for a multitude of biological processes, such as the attachment of viruses to the membrane of host cells. To scrutinize such (and other) processes in detail, it is inevitable to quantify the valency (i.e., the number of engaged single interactions) on the level of single viruses, which is still a major challenge as the individual interaction is typically weak and accordingly each interaction is maintained only transiently, causing temporal fluctuations of the valency. Hence, the quantification of multivalent interactions therefore requires methods that on the one hand allows for the characterization of many viruses in parallel (while keeping the single-virus resolution) but also provide, on the other hand, sufficient temporal resolution to follow temporal fluctuations of the valency.
In this context, we apply advanced microsopic techniques (e.g., total internal reflection fluorescence microsopy, confocal microsopy, fluorescence correlation spectroscopy) in order to extract the mobility of bilayer-linked bioparticles (e.g., viruses that are bound to their membrane receptors). The underlying rationale is motivated by the fact that the motion of a bioparticle with diameter < 400 nm is limited by the motion of the associated lipids but not its hydrodynamic size, implying that the bioparticle mobility decreases with increasing valency. Recently, we proved this hypothesis true for bilayer-linked liposomes and currently we extend this concept toward virus-receptor interactions, aiming to extract the"binding fingerprint" of this kind of multivalent interaction (i.e., the valency, on- and off-rate distributions) and how this fingerprint is modulated by application of virus inhibitors.
1. Block, S.* Brownian motion at lipid membranes: A comparison of hydrodynamic models describing and experiments quantifying diffusion within lipid bilayers. Biomolecules 8, 30 (2018).
2. Block, S.*; Zhdanov, V. P.; Höök, F.* Quantification of multivalent interactions by tracking single biological nanoparticle mobility on a lipid membrane. Nano Letters16, 4382-4390 (2016).
Optofluidic setups combine microfluidic channel architectures with optical microscopy, thereby allowing to characterize interfacial processes with high sensitivity and accuracy (using microscopy) in dependence of changes induced in the properties of the bulk solution (using microfluidics). We apply this concept in several projects by using the flow (passing through the channel) to create well-defined shear rates, which in turn create well-defined shear forces acting on interface-linked nanoparticles. In one set of experiments this shear force is used to load the linker, thereby allowing to perform shear force-based single-molecule force spectroscopy experiments (similar to those done using atomic force microscopy or optical/magnetic tweezers) yielding force-extension relationship of the linker or detachment kinetics of receptor-ligand interactions in dependence of the applied force. In another set of experiments we use the shear force to control the movement of bilayer-linked nanoparticles within the channel, thereby allowing to separate different species (e.g., transmembrane proteins or lipid receptors) within the bilayer based on their mobile properties.
In any case, since microscopy is used as readout, the fate of up to 1000 individual objects can be followed within a single experiement, thereby providing excellent measurement statistics even for short measurement times combined with the possibility to characterize heterogeneous samples. In our home-made setup we currently archieve sub nm resolution in the measurement of force-extension curves of polymers or proteins, while the applicable force ranges typically between few fN up to 10 pN. Currently, we use this tool to perform high-throughput single-molecule force spectroscopy experiments for various biophysical questions, e.g., the quantification of the binding strength of multivalent interactions close to equilibrium or for studies of the complex unfolding patterns of talin rod fragments.
1. Block, S.*; Johansson Fast, B.; Lundgren, A.; Zhdanov, V.P.; Höök, F.* Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity. Nature Communications 7, 12956 (2016).
2. Lundgren, A.O.#; Johansson Fast, B.#; Block, S.; Agnarsson, B.; Reimhult, E.; Gunnarsson, A.; Höök, F.* Affinity purification of membrane proteins in native supported membranes. Nano Letters 18, 381-385(2018).
Besides the above-mentioned projects, we are also interested in designing and applying new assays that allow for biophysical characterizations with single-virus, single-liposom, single-enzyme, or single-lipid resolution.These assays typically rely on advanced microscopy techniques (such as total internal reflection fluorescence microsopy, confocal microsopy, fluorescence correlation spectroscopy) that are used to characterize either the mobility of bilayer-associated compounds or the spectral properties of fluorescent dyes in order to extract the desired information. We used these approaches in the past
(i) to characterize lipid complexes formed by salt-mediated lipid-lipid interactions, based on measuring the complex mobility with high spatial and temporal resolution (< 20 nm, < 10 ms),
(ii) to quantify the turnover rate of single cytochrome bo3 ubiquinol oxidases after reconstitution in liposomes containing a pH-sensitive dye, and
(iii) to quantify size and content of bilayer-linked liposomes, based on measuring stochastic and deterministic components of their motion under shear flow.
Currently, we extend these assays to control, for example, the rate of electron injection into cytochrome bo3 ubiquinol oxidases using microfluidics or to study the penetration of single viruses through biohydrogels.
1. Block, S.*; Acimovic, S.S.; Länk, N.O.; Käll, M.*; Höök, F.* Antenna-enhanced fluorescence correlation spectroscopy resolves calcium-mediated lipid-lipid-interactions. ACS Nano 12, 3272-3279 (2018).
2. Berg, J.#; Block, S.#; Höök, F.; Brzezinski, P. Single proteoliposomes with E. coli quinol oxidase: proton pumping without transmembrane leaks. Israel Journal of Chemistry 57, 437-445 (2017).
3. Block, S.*; Johansson Fast, B.; Lundgren, A.; Zhdanov, V.P.; Höök, F.* Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity. Nature Communications 7, 12956 (2016)