Mode of Heavy Meromyosin Adsorption and Motor Function Correlated with Surface Hydrophobicity and Charge

Abstract

The in vitro motility assay is valuable for fundamental studies of actomyosin function and has recently been combined with nanostructuring techniques for the development of nanotechnological applications. However, the limited understanding of the interaction mechanisms between myosin motor fragments (heavy meromyosin, HMM) and artificial surfaces hampers the development as well as the interpretation of fundamental studies. Here we elucidate the HMM−surface interaction mechanisms for a range of negatively charged surfaces (silanized glass and SiO2), which is relevant both to nanotechnology and fundamental studies. The results show that the HMM-propelled actin filament sliding speed (after a single injection of HMM, 120 μg/mL) increased with the contact angle of the surfaces (in the range of 20−80°). However, quartz crystal microbalance (QCM) studies suggested a reduction in the adsorption of HMM (with coupled water) under these conditions. This result and actin filament binding data, together with previous measurements of the HMM density (Sundberg, M.; Balaz, M.; Bunk, R.; Rosengren-Holmberg, J. P.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Månsson, A. Langmuir 2006, 22, 7302−7312. Balaz, M.; Sundberg, M.; Persson, M.; Kvassman, J.; Månsson, A. Biochemistry 2007, 46, 7233−7251), are consistent with (1) an HMM monolayer and (2) different HMM configurations at different contact angles of the surface. More specifically, the QCM and in vitro motility assay data are consistent with a model where the molecules are adsorbed either via their flexible C-terminal tail part (HMMC) or via their positively charged N-terminal motor domain (HMMN) without other surface contact points. Measurements of ζ potentials suggest that an increased contact angle is correlated with a reduced negative charge of the surfaces. As a consequence, the HMMC configuration would be the dominant configuration at high contact angles but would be supplemented with electrostatically adsorbed HMM molecules (HMMN configuration) at low contact angles. This would explain the higher initial HMM adsorption (from probability arguments) under the latter conditions. Furthermore, because the HMMN mode would have no actin binding it would also account for the lower sliding velocity at low contact angles. The results are compared to previous studies of the microtubule−kinesin system and are also discussed in relation to fundamental studies of actomyosin and nanotechnological developments and applications.

Persistence Length of Human Cardiac α-Tropomyosin Measured by Single Molecule Direct Probe Microscopy

“…Using highly sensitive assays of function–in vitro motility assays–we have previously demonstrated that positively charged surfaces are good substrates for functional binding of another molecule (myosin or the two-headed, proteolytic digest fragment of myosin called heavy meromyosin, HMM) that has an α-helical coiled-coil tail [39], [40], [41]. To achieve functional binding of myosin (or HMM), the primary mode of surface adsorption must be through the α-helical coiled-coil tail region rather than the motor domains, or heads [42]. Furthermore, we initially tested a wider range of p-Lys concentrations, but chose 0.01% (w/v) as the minimum concentration at which we observed statistically sufficient numbers of clearly distinguishable structures consistent with single molecules of αTm (data not shown).…”

Translational actomyosin research: fundamental insights and applications hand in hand

“…Indeed, no later results have contradicted this idea (cf. Sundberg et al 2006a; Albet-Torres et al 2007, 2010, Persson et al 2010). When the silanization is performed in routine lab environments (ordinary fume hood without controlled environment as in a glove box) it generally leads to surfaces exhibiting similar contact angles with water droplets (similar wetting behavior) as nitrocellulose (advancing contact angles 70–80°; simply denoted “contact angles” below) but, not unexpectedly, considerably lower root-mean-square roughness (Albet-Torres et al 2007; Sundberg et al 2003; based on atomic force microscopy).…”

“…Systematic studies with the goal to understand the motor–surface interactions have therefore been performed both for the microtubule–kinesin system (Fischer and Hess 2007; Hiratsuka et al 2001; Kerssemakers et al 2006; Ozeki et al 2009) and for actomyosin (Albet-Torres et al 2007, 2010; Balaz et al 2007; Jaber et al 2003; Månsson et al 2008; Nicolau et al 2007; Persson et al 2010; Sundberg et al 2003, 2006a). …”

Mechanical Properties of Organelles Driven by Microtubule-Dependent Molecular Motors in Living Cells

“…In these experiments, motors are attached to artificial cargos such as glass or polystyrene beads by different protocols which include indirect linkers such as streptavidin-biotin [21] or direct adsorption to a surface treated with a blocking protein [22]. The interactions with the surface, the linkers and/or blocking molecules may affect the properties of the motor as was proposed to explain the different behavior of heavy meromyosin when attached to surfaces with different hydrophobicities [23].…”