Effects of Surface Adsorption on Catalytic Activity of Heavy Meromyosin Studied Using a Fluorescent ATP Analogue

Abstract

Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.

Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin

“…Whereas a small increase in the basal MgATPase activity cannot, per se , cause lower sliding velocity, the combination of reduced velocity and an increased basal MgATPase have been observed in response to several point mutations within the myosin motor domain (45–47). A similar combination of effects has also been observed by exchanging regular MgATP for a fluorescent MgATP analogue (48). The different effects of amrinone on HMM and S1 is considered in the supplemental material.…”

Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

“…However, extensive interactions between quantum dots attached to actin filaments and the TMCS-surface are unlikely at the high HMM incubation concentrations (50–120 µg/ml) used here. This is attributed to high HMM surface density [42], [43], [84] that would be expected to block access to the surface (cf. [83]).…”

In vitro and in vivo single myosin step-sizes in striated muscle

“…An alternative model has 3 microdomains on the surface that adsorb myosin and impose these 3, 5, and 8 nm step-sizes (the 3 step-size microdomain model) that is based on evidence suggesting that a functionalized (but not nitrocellulose coated) surface adsorbed myosin skeletal HMM has heterogeneous (modeled as 3 but probably more) rates of a myosin ATPase (step-size was not measured) (Balaz et al 2007). In this alternative model, changing step-frequencies observed with phosphorylation, OM binding, or the truncated version of the vELC are due to the different affinities these isoforms have for the 3 step-size microdomains.…”