A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis

Abstract

There has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.

Keywords: 10S myosin; Phalloidin; Protein purification; Smooth muscle myosin 2; Transmission electron microscopy.

Fig. 1

Workflow of the preparation and analysis of Myosin-II from chicken gizzard tissues using phalloidin. Details of the buffers and other conditions have been described in the “Materials and methods” section

Fig. 2

Graphical illustration of the myosin-II purification using phalloidin. Conceptual interpretation of the reactions undertaking at the molecular level during the course of purification. Every step is a consequence of the treatment or condition applied in the previous step

Fig. 3

TEM analysis of myosin 10S molecules. a Myosin obtained from the supernatant post phalloidin treatment was diluted with ATP, MgCl₂ low salt buffer and cross-linked with 0.1% glutaraldehyde to a final concentration of 100 nM. Black arrows show the head region of the folded myosin molecule. Scale bar, 100 nm. b Alignment and classification of the particles obtained from micrographs was done with 2500 compact particles using SPIDER. The head aligned images of the class averages show the general variance in the orientation of the head and tail. Scale bar, 20 nm

Fig. 4

Step-wise SDS-PAGE and densitometric analysis of smooth muscle myosin-II purification. The proteins were electrophoresed on a 4-20 % gradient gel stained by Coomasie brilliant blue. (S) and (P) demonstrate supernatant and pellet, respectively collected during the washing a, extraction and co-sedimentation with or without phalloidin b steps of the purification after centrifugation. c Comparative densitometric analysis myosin and actin contents between the supernatants of the original actomyosin extract and with (PHD), or without (CONT) phalloidin treatment. d SDS-PAGE profile of eluted fractions from myosin purification using short size exclusion chromatography. S indicates the phalloidin treated supernatant; lanes 1-13 represent eluted fractions. Note that the bands representing Myosin Heavy Chains (HC), Regulatory Light chains (RLC) and Essential Light chains (ELC) and Actin (Ac) have been indicated by arrowheads respectively. The pre-stained protein markers were represented as (M). Note (EXT) designates actomyosin original extract