Alternative Names for alpha-actinin

1. Actinin alpha 1
2. ACTN 1
3. ACTN1
4. Alpha Actinin 1
5. Alpha actinin cytoskeletal isoform
6. F actin cross linking protein
7. FLJ40884
8. Non muscle alpha actinin 1

Force Measurement (1)

The function of muscle is to exert force and to do mechanical work. It would therefore be highly desirable to be able to measure the force exerted upon single thin filaments by thick filaments (myosin).

In the normal in vitro motility assay the thin filaments are unloaded, therefore the interpretation of the results obtained from such systems is limited to comparisons with unloaded shortening in intact muscle.

The principle of novel in vitro motility assay is to place an internal load upon the thin filament to retard filament movement due to the myosin motor. This is achieved by using an actin-binding protein attached to the cover glass along with the immobilized myosin motor protein. The greater the force on an actin filament, the higher the concentration of actin-binding protein needed to stop movement.

Bing, W., A. Knott, and S.B. Marston, A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments. Biochem J, 2000. 350 Pt 3: p. 693-9

Hill Equation

The Hill equation is an equation used in enzyme characterization. In biochemistry, the binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule (this is known as Cooperative binding).

The Hill Coefficient (n) describes the fraction of the enzyme saturated by ligand as a function of the ligand concentration; it is used in determining the degree of cooperativity of the enzyme.

1. Positively cooperative reaction (n > 1): Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules increases.
2. Negatively cooperative reaction (n < 1): Once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules decreases.
3. Noncooperative reaction (n = 1): The affinity of the enzyme for a ligand molecule is not dependent on whether or not other ligand molecules are already bound.

Velocity/Force-pCa Relation

Calcium sensitivity (pCa50): negative log of the calcium concentration at half-maximal velocity / force) of thin filament;

Cooperativity (Hill Coefficient): Values are expressed as least-square regression of the fit to the Hill equation ± SE;

Maximal activation (Vmax or Fmax): maximal calcium-activated velocity / of thin filament.

VanBuren, P., et al., Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system. Am J Physiol Heart Circ Physiol, 2002. 282(5): p. H1665-71

Reconstitution of Thin Filaments

Reconstitution of thin filaments from actin, troponin, and tropomyosin was done by mixing the proteins at a concentration of 2 µM F-actin, 0.5 µM tropomyosin, and 0.48-0.5 µM troponin in F-actin buffer (4 mM imidazole, pH 7.1 at 25C), 2 mM MgCL2, 0.5 mM ATP, 3 mM NaN3, 1 mM DTT). The solution was allowed to incubate overnight before use.

Homsher, E., et al., Calcium regulation of thin filament movement in an in vitro motility assay. Biophys J, 1996. 70(4): p. 1881-92

Thin filaments were then labeled with rhodamine-phalloidin at a 1:1 actin-to-phalloidin ratio in low-salt buffer (in mM: 25 KCl, 25 imidazole, 5 MgCL2, 10 DTT, and 2 EGTA, pH 7.4) and stored overnight a 4°C before use in the in vitro motility assay.

VanBuren, P., et al., Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system. Am J Physiol Heart Circ Physiol, 2002. 282(5): p. H1665-71

Variability in Motor Velocity

The wide velocity variations observed are rather surprising and cannot be explained by simple kinetics models. A simple class of models applied to many motors assumes that discrete stepping is tightly coupled to the ATP hydrolysis cycle, occurring at a well defined average rate. In such a model, velocity variations occur because Brownian fluctuations play an important role in driving the motor transitions. In the most random possible case, referred to as a ‘‘Poisson stepper,’’ one expects an exponential distribution of waiting times between steps. …

We emphasize that two types of variability were observed: (i) variation in the average velocities of different complexes, referred to in the literature as ‘‘static disorder,’’ and (ii) variations in the velocities of single complexes in time, referred to as ‘‘dynamic disorder’’ . Although it is difficult to rule out protein degradation or instability as a contributing factor to static disorder, such effects cannot fully explain dynamic disorder because the velocity was observed to vary both up and down in time. Degradation would be expected to only cause decreases.

…, Similar levels of static and dynamic disorder in enzyme kinetics have been reported in several previous single-molecule experiments… Our data show that such behavior can also occur in a more complex, multicomponent motor. Other researchers have provided evidence that such variability can be attributed to the existence of multiple active conformational states of the enzyme complexes and slow interconversion between them. …, suggest multiple … conformers and assembly states exhibiting different levels of ATPase activity.

Fuller, D.N., et al., Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability. Proc Natl Acad Sci U S A, 2007. 104(43): p. 16868-73

The NH2-terminal Segment of TnT

TnT, the tropomyosin (Tm) binding subunit of the troponin (Tn) complex, contains a globular COOH-terminus and a long NH₂-terminal tail. The globular portion of TnT binds to troponin C (TnC; the calcium binding subunit), troponin I (TnI; the inhibitory subunit), and Tm in a calcium-dependent manner.

The NH₂-terminal segment of TnT (TnT-NH₂⁺)highly charged, and binds at the NH₂-terminal/COOH-terminal overlap of two adjacent Tm molecules in a calcium-independent manner, thus providing a tether for the entire Tn complex to the thin filament during muscle activation.

The TnT-NH₂⁺enhances the binding of the Tm to actin and thus likely facilitates the communication of movement between adjacent Tm with thin filament activation. The overlap of adjacent Tm is felt to be a critical component of thin filament-mediated cooperative activation.

VanBuren, P., et al. Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system. Am J Physiol Heart Circ Physiol, 2002. 282 (5): p. H1665-71.