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Langmuir. 2015 Jun 9;31(22):6145-53. doi: 10.1021/acs.langmuir.5b01735. Epub 2015 May 27.

Molecular-Level Insights into Orientation-Dependent Changes in the Thermal Stability of Enzymes Covalently Immobilized on Surfaces.

Author information

1
†Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
2
§Department of Biophysics, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
3
‡Department of Macromolecular Science and Engineering, University of Michigan, 2300 Hayward Street, Ann Arbor, Michigan 48109, United States.
4
∥Department of Biological Chemistry, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, United States.

Abstract

Surface-immobilized enzymes are important for a wide range of technological applications, including industrial catalysis, drug delivery, medical diagnosis, and biosensors; however, our understanding of how enzymes and proteins interact with abiological surfaces on the molecular level remains extremely limited. We have compared the structure, activity, and thermal stability of two variants of a β-galactosidase attached to a chemically well-defined maleimide-terminated self-assembled monolayer surface through a unique cysteinyl residue. In one case the enzyme is attached through an α helix and in the other case through an adjacent loop. Both enzymes exhibit similar specific activities and adopt similar orientations with respect to the surface normal, as determined by sum-frequency generation and attenuated total reflectance FT-IR spectroscopies. Surprisingly, however, the loop-tethered enzyme exhibits a thermal stability 10 °C lower than the helix-tethered enzyme and 13 °C lower than the enzyme in free solution. Using coarse-grain models, molecular dynamics simulations of the thermal unfolding of the surface-tethered enzymes were able to reproduce these differences in stability. Thus, revealing that tethering through the more flexible loop position provides more opportunity for surface residues on the protein to interact with the surface and undergo surface-induced unfolding. These observations point to the importance of the location of the attachment point in determining the performance of surface-supported biocatalysts and suggest strategies for optimizing their activity and thermal stability through molecular simulations.

PMID:
25973638
DOI:
10.1021/acs.langmuir.5b01735
[Indexed for MEDLINE]

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