Myosin subfragment-1 is fully equipped with factors essential for motor function

Area of Science:

• Muscle contraction
• Biochemistry
• Molecular motors

Background:

• Myosin subfragment-1 (S1) is crucial for muscle contraction, interacting with actin filaments.
• Previous studies faced limitations in controlling S1 binding orientation, affecting measured velocities.
• Specific binding strategies are needed to accurately assess S1's intrinsic motor capabilities.

Purpose of the Study:

• To investigate the sliding velocity of actin filaments propelled by chicken skeletal myosin subfragment-1 (S1).
• To achieve specific binding of the S1 tail end to a surface for controlled experiments.
• To determine if S1 can generate normal fast movement and force when specifically bound.

Main Methods:

• Replaced the regulatory light chain (RLC) of S1 with a fusion protein (biotin-dependent transcarboxylase [BDTC]-chicken gizzard smooth muscle RLC [cgmRLC]).
• Attached the modified S1 to a biotin-coated glass surface via a biotin-avidin system for specific tail-end binding.
• Measured actin filament sliding velocity and actin-activated Mg-ATPase activity at 29 degrees C.

Main Results:

• The specifically bound S1 propelled actin filaments at a velocity of 6.8 ± 0.6 microm/sec.
• This velocity was 3.5-fold greater than previously observed direct binding (1.9 ± 0.3 microm/sec).
• The actin-activated Mg-ATPase activity remained similar to unmodified S1, and the velocity matched native chicken skeletal myosin.

Conclusions:

• Specific tail-end binding of S1 to a surface allows for measurement of its intrinsic fast movement capabilities.
• Myosin S1, when properly oriented, can produce normal fast actin filament movement.
• The results confirm S1's ability to hydrolyze ATP, generate force, and propel actin filaments efficiently.