Differential adsorption of variants of the Thermomyces lanuginosus lipase on a hydrophobic surface suggests a role for local flexibility.
Lipases are activated at interfaces between aqueous and hydrophobic phases, where they typically undergo conformational changes leading to significant activity increase. Here I use a quartz crystal microbalance with dissipation (QCM-D) to study changes in layer thickness and viscosity during the adsorption of variants of the Thermomyces lanuginosus lipase (TlL) onto a methyl-terminated hydrophobic surface. Unlike wildtype TlL, the variant Mut1, which shows improved performance under certain test conditions, shows a large dissipation increase during the binding process, leading to a significantly thicker layer. This altered adsorption behaviour may be linked to Mut1's changes in secondary structure. This is corroborated by the fact that four other TlL mutants with unaltered secondary structure showed wildtype-like absorption behaviour. Unlike wildtype TlL and the other variants, Mut1 contains several consecutive basic residues introduced into the C-terminal region which is close in space to the N-terminal part of the protein, which also contains several basic residues. Electrostatic repulsion between these two regions leading to local structural flexibility may facilitate altered adsorption behaviour and ultimately to improved enzymatic performance on a solid surface. QCM-D thus provides a good approach to screen protein variants for their adsorption properties on hydrophobic surfaces.
Otzen
Daniel
D
Interdisciplinary Nanoscience Centre, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. dao@inano.dk
D000327:Adsorption; D001203:Ascomycota / Q000201:enzymology*; D014644:Genetic Variation; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000493:pharmacokinetics*; D008954:Models, Biological; D011791:Quartz / Q000737:chemistry; D055672:Static Electricity; D013499:Surface Properties; D014783:Viscosity
D011791:Quartz; D008049:Lipase