The actin-myosin motor system plays important roles in cellular processes. In addition, actin and myosin have been used for developments towards nanotechnological applications in recent years. Therefore, fundamental biophysical studies of actin and myosin and the actomyosin force generating cycle are important both in biology and for nanotechnology where the latter applications require methodological insights for optimization. This dual goal is central in the present thesis with major focus on factors that control the function (e.g. velocity) and the effectiveness of transport of filaments (e.g. filament flexural rigidity) through nanoscale channels with supplementation of methodological insights. The thesis thus provides evidence that actin is a dynamic filament whose flexural rigidity is different at different MgATP concentrations as well as in the presence or absence of myosin binding. Furthermore, probing the myosin ATPase cycle with the myosin inhibitor blebbistatin revealed that velocity is easily modified by this drug. Our detailed studies also suggest that actin-myosin force generation is preceded by Pi release and that blebbistatin changes the rate limiting transition in the cycle from the attachment step to a step between weakly attached states. The studies of actin dynamics and of the actomyosin force generating cycle were largely performed using in vitro motility assay (IVMA) where surface adsorbed myosin motor or its proteolytic fragments propel fluorescently labeled actin filaments. The IVMA is often taken as the basis for developments towards different nanotechnological applications. However, in the IVMA, actomyosin motility is often negatively affected by the presence of “dead”, non-functional myosin heads. Therefore, in this thesis, two popular methods, that are often used to remove dead myosin heads, are analyzed and compared. It was found that after affinity purification, the in vitro actin sliding velocity is reduced compared to the control conditions, something that was not seen with the use of blocking actin. Therefore, the effects of the affinity purification method should be considered when interpreting IVMA data. This is important while using IVMA both for fundamental studies and for nanotechnological applications. Another issue in the use of IVMAs in nanotechnological applications is the requirement for expensive and time-consuming fabrication of nanostructured devices. We therefore developed a suitable method for regenerating molecular motor based bionanodevices without a need to disassemble the flow cell. Evidence is presented that, use of proteinase K with a suitable detergent (SDS or Triton X100) lead to successful regeneration of devices where both actin-myosin and microtubule-kinesin motility are used. Lastly, this thesis presents efforts to immobilize engineered light sensitive myosin motors on trimethyl chlorosilane (TMCS) derivatized surfaces for light operated switching of myosin motor in order to control actin movement in nano-networks. This has potential for developing a programmable junction in a biocomputation network. In brief, the described results have contributed both to the fundamental understanding of actin and myosin properties and the actomyosin interaction mechanisms. They have also given technical insights for molecular motor based bionanotechnology.

There exist complex mathematical problems that are important in real world applications such as weather prediction, molecular modelling, network route optimization and more. In general, such problems are solved using supercomputers with higher computing efficiency but this also consumes high energy along with high production and maintenance cost. Network-based biocomputation (NBC) is an alternate computing approach, now at development stage, that can perform parallel computing in a highly energy efficient manner. Actin and myosin constitute one type of molecular motor system that has been utilized for the development of NBC. These proteins are key components in the sarcomere, the smallest functional unit of muscle and their interactions that underlie muscle contraction are powered by the cellular fuel adenosine triphosphate (ATP). To solve larger complex problems using actin-myosin based NBC, factors such as maintained biological function and longevity of operation are essential for practical relevance. In this thesis, the in vitro motility assay (IVMA) has been used as a central method to study actomyosin function and its operation within NBC devices. In the IVMA, actin filaments are propelled by myosin motors that are immobilized on functionalized surfaces in a flow cell. With the aim to improve motile fraction by reducing the interaction between actin and non-functional motor heads in the IVMA, two known methods were quantitatively compared in paper I, the affinity purification and the blocking actin method. Both approaches significantly improved the motile fraction to above 90% but affinity purification, due to the presence of ATP during incubation, induced significant reduction in sliding velocity, not seen with blocking actin. In paper III, critical parameters in the actomyosin IVMA system were investigated allowing us to extensively improve function and longevity, including: biocompatibility of flow cell components, effects of air exposure with oxygen scavenging and nanofabrication parameters such as plasma etching type and time, process of resist development, and surface silanization time. The above developments together with optimized network encoding of the problems enabled us (paper IV) to solve four instances of 3-SAT problem encoded in NBC with 99% probability of satisfiability. In parallel, (paper II) a method have been developed to recycle the surfaces with immobilized motor proteins by treatment of proteinase-K enzyme and detergent. This will allow re-cycling of advanced NBC chips. Finally, with aim to develop programmable gating for NBC, attempts have been made towards the integration of engineered light sensitive myosin XI motors with nanofabricated devices made up of Au/SiO₂, SiO₂/polymer and glass/polymer (paper V). In addition important factors such as standardized motor density, limiting of air exposure and longevity function have been optimized in the use of light sensitive motors.

Overall, this thesis reports critical insights for the upscaling of actomyosin based NBC. Described results, are also useful for the development of actomyosin based nanotechnological applications such as biosensing or diagnostics and other fundamental studies based on single molecule or drug testing.