Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments

Area of Science:

• Biochemistry
• Structural Biology
• Actin Dynamics

Background:

• F-actin exhibits structural connectivity between the DNase I binding loop (residues 38-52) and the C-terminus.
• Understanding these interactions is crucial for deciphering actin's functional mechanisms.

Purpose of the Study:

• To investigate the structural connectivity between actin loop 38-52 and the C-terminus in F-actin.
• To determine the effect of copper binding on these structural interactions.

Main Methods:

• Fluorescence spectroscopy using dansyl ethylenediamine (DED) labeled actin.
• Proteolytic digestion with subtilisin and trypsin.
• Copper binding assays.
• Analysis of actin copolymers.

Main Results:

• Copper binding to Cys-374 in F-actin quenched DED fluorescence at Gin-41, indicating proximity.
• N-ethylmaleimide labeling of Cys-374 blocked copper binding and abolished quenching.
• Copper binding increased subtilisin cleavage at loop 38-52 in F-actin but not G-actin.
• Acto-myosin subfragment-1 ATPase activity and tryptic digestion sites were unaffected by copper.

Conclusions:

• Copper binding to Cys-374 induces intermolecular coupling between loop 38-52 and the C-terminus in F-actin.
• This perturbation is due to altered intermolecular contacts, not extensive structural changes in actin monomers.
• Copper binding specifically affects the loop 38-52 environment within the F-actin polymer.