Abstract
The processive motor myosin V has a high affinity for actin in the weak binding states when compared with non-processive myosins. Here we test whether this feature is essential for myosin V to walk processively along an actin filament. The net charge of loop 2, a surface loop implicated in the initial weak binding between myosin and actin, was increased or decreased to correspondingly change the affinity of myosin V for actin in the weak binding state, without changing the velocity of movement. Processive run lengths of single molecules were determined by total internal reflection fluorescence microscopy. Reducing the net positive charge of loop 2 significantly decreased both the affinity of myosin V for actin and the processive run length. Conversely, the addition of positive charge to loop 2 increased actin affinity and processive run length. We hypothesize that a high affinity for actin allows the detached head of a stepping myosin V to find its next actin binding site more quickly, thus decreasing the probability of run termination.
Highlights
We previously showed that replacing loop 2 of mouse myosin V with the corresponding loop from a yeast class V myosin (Myo4p, net loop charge of 0) caused a dramatic reduction in processive run lengths, especially at high ionic strength [20]
Myosin V goes through an intermediate state in which the rear head is strongly attached to actin, and the lead head undergoes a diffusive search for the actin binding site [4, 5, 21]
We propose that a high affinity for actin ensures that the lead head finds the binding site faster, reducing the lifetime of the vulnerable singly bound intermediate and increasing processive run lengths
Summary
Myosin V Constructs—The myosin V constructs used in this study were engineered to contain a biotin tag at the C terminus for attachment to streptavidin-coated quantum dots (Qdots) (Invitrogen). The wild-type (WT) murine myosin V HMM construct was truncated at amino acid 1098 followed by an 88-amino-acid sequence segment from the Escherichia coli biotin carboxyl carrier protein [22, 23] and a FLAG tag to facilitate purification. During expression in Sf9 cells, the biotin carboxyl.
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