Role of Cel5H protein surface amino acids in binding with clay minerals and measurements of its forces

Applied Microscopy

Han Dae Yun¹  Palaksha K. Javaregowda²  Renukaradhya K. Math³  Nagakumar Bharatham⁴ 

[1] Division of Applied Life Sciences, Gyeongsang National University, 660701, Chinju, Republic of Korea;SDM Research Institute for Biomedical Sciences, 5th Floor, Manjushree Building, SDM College of Medical Sciences & Hospital Campus, Shri Dharmasthala Manjunatheshwara University, 580009, Dharwad, Sattur, India;SDM Research Institute for Biomedical Sciences, 5th Floor, Manjushree Building, SDM College of Medical Sciences & Hospital Campus, Shri Dharmasthala Manjunatheshwara University, 580009, Dharwad, Sattur, India;Division of Applied Life Sciences, Gyeongsang National University, 660701, Chinju, Republic of Korea;The University of Trans-Disciplinary Health Sciences and Technology (TDU), 560064, Bengaluru, Karnataka, India;

关键词: Clay mineral;    Protein binding;    Homology modeling;    Mutation;    AFM;    Adhesion force;   

DOI  :  10.1186/s42649-021-00066-7

来源: Springer

【 摘 要 】

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

【 授权许可】

CC BY