Probing Changes in Ca2+-Induced Interaction Forces between Calmodulin and Melittin by Atomic Force Microscopy.

Sheng Huang,Jianhua Wang,Yuna Fu,Heng Sun,Yan Wang

doi:10.3390/mi11100906

Abstract

Mechanobiology studies the means by which physical forces and mechanical properties change intra- or inter- biological macromolecules. Calmodulin (CaM) is involved in physiological activities and various metabolic processes in eukaryotic cells. Although the configuration changes in the interaction between calmodulin and melittin have been studied, the biomechanical relationship of their interaction has rarely been explored. Here, we measured the adhesion forces between calmodulin and melittin in solutions of gradient concentration of calcium ions using atomic force microscopy (AFM). We found that the specific (Fi) and nonspecific (F0) adhesion forces between single melittin and calmodulin in a PBS solution were 69.4 ± 5.0 and 29.3 ± 8.9 pN, respectively. In the presence of 10−7 to 10−3 M Ca2+ PBS solution, the Fi increased significantly to 93.8 ± 5.0, 139.9 ± 9.0, 140.4 ± 9.7, 171.5 ± 9.0, and 213.3 ± 17.8 pN, indicating that the unbinding force between melittin and calmodulin increased in the presence of Ca2+ in a concentration-dependent manner. These findings demonstrated that biomechanical studies based on AFM could help us better understand the melittin/calmodulin-binding processes in the presence of calcium and help us design and screen peptide drugs based on calmodulin.

Highlights

Mechanobiology is an emerging research field with the primary purpose of linking biological and mechanical engineering, which has attracted increasing attention and participation from researchers in recent years [1,2]
The reason is that protein-based receptor–ligand interactions [6,7,8] and protein folding [9,10] activities are known to run through the entire biological processes, and changes in the mechanical properties of these processes play a crucial role [11,12,13]
The results showed that calcium ions significantly affected the mechanical interaction between calmodulin and melittin

Summary

Introduction

Mechanobiology is an emerging research field with the primary purpose of linking biological and mechanical engineering, which has attracted increasing attention and participation from researchers in recent years [1,2]. Many technologies have emerged to explore the interaction between or within biological macromolecules, such as biomembrane force probes (BFPs) [14,15], optical tweezers [16,17], microneedle manipulation [18,19], magnetic tweezers [20,21], and atomic force microscopy (AFM) [22,23,24] Among these evolving technologies, AFM has shown increasing advantages due to: (1) the fact that it can be operated with high force sensitivity and obtained nanometric morphology resolution [25,26]; (2) its ability to simulate the measurement of intermolecular or intramolecular interactions under physiological conditions, such as receptor–ligand interactions and protein folding, which might help explain some essential pathogenic mechanisms [27,28]; (3) its Micromachines 2020, 11, x. To further study the structure–activity relationship between melittin and calmodulin, research on their interaction forces, whether in the absence or presence of calcium ions, should be conducted because the protein–ligand interaction force plays a critical role in the biological system processes. The specific interaction force between melittin and calmodulin increased with the increase of the calcium concentration

Materials

Preparation of Gold Substrate

Protein Immobilization onto the Gold Surfaces

Measurement of the Melittin-Calmodulin Adhesion Force

Conclusions