Masatoshi Morimatsu

Observation of Dynamical Conformational Changes of Skeletal Muscle Actin Filament

Actin is a ubiquitous and essential protein that has a primary role in a number of important cellular phenomenon including muscle contraction, cell motility, and cell division. It has two forms, monomeric G-actin and filamentous F-actin, which are determined by the cellular environment. Understanding the dynamic conformations between the two is important for understanding actins cellular functions, yet only recent advancements have been made. Oda and Maeda used X-ray fiber diffraction to describe the multiple conformations of F-actin. To investigate the dynamics of these F-actin states, we here describe the preparation of fully active α-actin obtained from a baculovirus expression system suitable for single molecule FRET (smFRET) measurements. We developed α-actin recombinants in which two domains of the tetramers have specific sites for fluorescent probes. Actin filaments showed different dynamic conformational changes between these domains in the presence and absence of myosin S1. Specially, we observed several sequential transition states on a second or sub-second order. This technique, which combines specific labeling and smFRET, offers to significantly expand the information acquired in actin studies.

Multiple Structural Forms of Actin in the Filamentous State

One of the most abundant proteins in eukaryotes is actin, a ubiquitous protein that plays a role in cell dynamics like cell migration. The dynamics of actin filament treadmilling is regulated by two actin structural states: globular actin (G-actin) and filamentous actin (F-actin). While G-actins crystal structure has been solved by several groups, F-actins has not. Although recently it was reported that the structure for the two actins differ (Oda et al), there is still much to resolve on the matter of their dynamic structures. Here we observed the dynamics of the actin structural states under various conditions by using single-molecule FRET in combination with total internal reflection fluorescence microscopy. To conduct these experiments, we first labeled actin residues 41 and 374, having substituted Gln 41 with Cys. The new Cys 41 site along with Cys 374 were used for site-directed labeling by SH-group reactive fluorescent dyes. We found that F-actin has at least two distinct states, and that the population distribution of these states was dependent on the ionic conditions. We are currently investigating these states by performing FRET measurements for observing the long -time transition between the two states.