In vitro assays of molecular motors - impact of motor-surface interactions

Abstract

In many types of biophysical studies of both single molecules and ensembles of molecular motors the motors are adsorbed to artificial surfaces. Some of the most important assay systems of this type (in vitro motility assays and related single molecule techniques) will be briefly described together with an account of breakthroughs in the understanding of actomyosin function that have resulted from their use. A poorly characterized, but potentially important, entity in these studies is the mechanism of motor adsorption to surfaces and the effects of motor surface interactions on experimental results. A better understanding of these phenomena is also important for the development of commercially viable nanotechnological applications powered by molecular motors. Here, we will consider several aspects of motor surface interactions with a particular focus on heavy meromyosin (HMM) from skeletal muscle. These aspects will be related to heavy meromyosin structure and relevant parts of the vast literature on protein-surface interactions for non-motor proteins. An overview of methods for studying motor-surface interactions will also be given. The information is used as a basis for further development of a model for HMM-surface interactions and is discussed in relation to experiments where nanopatterning has been employed for in vitro reconstruction of actomyosin order. The challenges and potentials of this approach in biophysical studies, compared to the use of self-assembly of biological components into supramolecular protein aggregates (e.g. myosin filaments) will be considered. Finally, this review will consider the implications for further developments of motor-powered lab-on-a-chip devices.

Translational actomyosin research: fundamental insights and applications hand in hand

“…As a result of this mode of adsorption, the heads may extend appreciably away from the surface (Albet-Torres et al 2010; Persson et al 2010). This configuration is believed to completely dominate on moderately hydrophobic surfaces (TMCS) whereas the available evidence is consistent with HMM adsorption to pure glass/SiO 2 , preferably via positively charged loops in the actin binding region (Albet-Torres et al 2010; Månsson et al 2008; Persson et al 2010). This idea is consistent with high-speed atomic force microscopy images (Ando et al 2001a; for movies see Ando et al 2001b) where myosin V is imaged while adsorbed to freshly cleaved mica[a silicate mineral with, generally low contact angle and negative surface charge (Yang et al 2007)].…”

“…2). (Albet-Torres et al 2010; Månsson 2010; Månsson et al 2008; Sundberg et al 2006a; Vikhoreva and Månsson 2010). In contrast, the total HMM density on the different surfaces did not appear to differ appreciably.…”

“…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). …”

Long-Term Storage of Surface-Adsorbed Protein Machines

“…(38) With regard to HMM, in particular, it has been indicated that freezing in solution at −20 °C for later use in the IVMA is not advisible, even if glycerol is added as a cryoprotecting agent. 36,39 This may be understood because some regions of the HMM molecule exhibit conformational instability (cf.ref (6)) whereas other regions are sensitive to oxidation. The good preservation of function in the present work may, to a substantial degree, be attributed to factors related to the surface immobilization of the proteins.…”

“…The actomyosin motor system relies on intact enzymatic function, actin polymerization, and actomyosin bimolecular interactions, and it is sensitive to deterioration by several mechanisms. 4−6 It should therefore be a critical test system for investigations of functional changes during storage.…”