Chunmei Lv1, Xiang Gao1, Wenfei Li1, Bo Xue2, Meng Qin1, Leslie D. Burtnick3, Hao Zhou4, Yi Cao1,*, Robert C. Robinson2,5,* & Wei Wang1,*
1 National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, 22 Hankou Road, Nanjing 210093, China.
2 Institute of Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, Singapore 138673, Singapore.
3 Department of Chemistry and Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
4 State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China.
5 Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
* Corresponding authors
Published in Nature Communications on 07 August 2014
Force is increasingly recognized as an important element in controlling biological processes. Forces can deform native protein conformations leading to protein-specific effects. Protein–protein binding affinities may be decreased, or novel protein–protein interaction sites may be revealed, on mechanically stressing one or more components. Here we demonstrate that the calcium-binding affinity of the sixth domain of the actin-binding protein gelsolin (G6) can be enhanced by mechanical force. Our kinetic model suggests that the calcium-binding affinity of G6 increases exponentially with force, up to the point of G6 unfolding. This implies that gelsolin may be activated at lower calcium ion levels when subjected to tensile forces. The demonstration that cation–protein binding affinities can be force-dependent provides a new understanding of the complex behaviour of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.
Figure Legend: Mechanical properties of gelsolin domain (G6) probed by single-molecule AFM. (a) Cartoons highlighting the initial activation stages of gelsolin. Occupation of any of the homologous calcium-binding sites (white circles) by a calcium ion will begin the activation process that drives conformational reorganization and concomitantly creates inter-domain strain. The resulting inter-domain strain will increase the affinity of G6 for calcium, resulting in calcium ion binding to G6 and the release of the C-terminal tail (purple), which in turn exposes the F-actin-binding site on domain 2. (b, c) The structure of G6 in the absence and presence of Ca2+ (black sphere). (d) A schematic of the hetero-polyprotein (GB1–G6)4 used for single-molecule force spectroscopy experiments. (e) Typical set of approaching (grey line) and retraction traces for mechanical unfolding of calcium-free apo or calcium-bound holo forms of (GB1–G6)4. (f) Unfolding force histograms of apo and holo G6. (g) Pulling speed dependency of apo and holo G6.
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