Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

Abstract

The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.

Correlation of Molecular and Functional Effects of Mutations in Cardiac Troponin T Linked to Familial Hypertrophic Cardiomyopathy

“…It has been suggested that thin filament FHC mutant proteins (such as the Arg-92 hot spots) markedly alter the nature of the interaction between actin and myosin (16). Consequently, the changes in cooperativity observed in the Arg-92 hot spots may be indicative of alterations in the allosteric regulation of Tm by Tn, whereby the cooperative interactions of Tn-Tm may produce structural changes allowing for strong actin-myosin binding (the transition to "open" state in the three-state model of myofilament activation) (14).…”

“…Regulated in Vitro Motility Assay-Motility assay procedures and measurement were performed as described previously (14). In brief, a nitrocellulose-coated coverslip was attached to a glass microscope slide using double-faced tape, forming a flow cell chamber.…”

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component, Troponin T

“…The dialyzed Tn and Tm were then used to produce reconstituted thin filaments (RTF) under rigor conditions (i.e. in the absence of ATP) within the flow cell used for the motility assays (41).…”

“…Following this step, 0.35 M Tm and 0.75 M Tn were added and allowed to incubate in the flow cell for 7 min, resulting in the formation of RTF. This method, which had been used previously, creates RTF capable of fully regulating the actomyosin interaction, as evidenced by the lack of filament movement at pCa levels above 9, while also showing a strong Ca 2ϩ -dependent increase in velocity, from pCa ϳ7 to 4 (41). A solution containing 100 nM excess Tn and Tm was also added to the final assay buffer to ensure full and complete activation regulation of the RTFs as described previously (41,44).…”

“…This method, which had been used previously, creates RTF capable of fully regulating the actomyosin interaction, as evidenced by the lack of filament movement at pCa levels above 9, while also showing a strong Ca 2ϩ -dependent increase in velocity, from pCa ϳ7 to 4 (41). A solution containing 100 nM excess Tn and Tm was also added to the final assay buffer to ensure full and complete activation regulation of the RTFs as described previously (41,44). The motion of the labeled regulated actin filaments was visualized and recorded using a Nikon Ti-U inverted microscope, with a 100ϫ, 1.4 NA oil-coupled objective with a thermometer (20/20 Technologies, Wilmington, NC) to maintain the temperature at 30.0°C for all experiments.…”