Study for mechanical properties of an actin-gelation protein, ABP-280 by the single-molecule manipulation

2000 Fiscal Year Final Research Report Summary

• Project/Area Number: 11680656
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: ITO Tadanao Kyoto University, School of Science, Associate Professor, 理学研究科, 助教授 (90093187)
• Co-Investigator(Kenkyū-buntansha): YAMAGAKI Masakagu Shiguoka University, Faculty of Science, Associate Professor, 理学部, 助教授 (70200665) ; OHASHI Kagup Chiba University, Faculty of Science, Professor, 理学部, 教授 (90114248)
• Project Period (FY): 1999 – 2000
• Keywords: actin gel / ABP-280 / force-extersion curve of single molecules / AFM / rheology / cytoskeleton / cell motility / mechanical response of cell

Research Abstract

ABP -280 is a dimeric protein with equivalent subunits of rod-like structure. The two 280 kDa subunits associate with each other through interchain hydrophobic interaction near the carboxyl-terminus, and bind to F-actin near the amino-terminus of the subunit. The remaining 90% of the sequence comprises 24 repeats in line, each - 96 residues long, predicted to have stretches of anti-parallel β-sheets of the immunogloblin (Ig) -like motifs.
ABP-280 crosslinks F-actin filaments, making a three dimensional orthogonal actin gel at a molar ratio to actin monomer less than 1/500 (actin/ABP gel). The gel structure is similar to that of actin cytoskeleton in cortex of cell.
In the present study, we have investigated rheological properties of actin/ABP gels by an oscillation-type rheometer and mechanical properties of single ABP-280 molecules by an atomic force microscopy (AFM), and obtained the following results.
(1) Actin/ABP gels behave as "elastic body" under shear strain less than 20%, as "visco-elastic body" from 20% to 100% which can change the elasticity according to the degree of shear strain, and as viscous fluid over 100%. Moreover, the gel can recover its original structure after released from the shear strain over 100%.
(2) When single ABP-molecules are pulled by AFM, the individual repeating units are sequentially unfolded with the pulling forces of 50〜200 pN, finally extended as 10 times long as the original one. The molecules once extended are completely refolded in a few seconds after the pulling forces are removed off.
We may compare such a function of ABP-280 to a kind of spring hinge, spring constant of which is adjusted variably to the shear strain by reversible unfolding/refolding transition in the repeating units, endowing the gels with ability to recover the original structure after released from a large strain.