Enzymatic Proteolysis of a Surface-Bound α-Helical Polypeptide
| Autor: | Curtis W. Frank, James T. Kellis, Luis G. Cascao-Pereira, Channing R. Robertson, Jasper O. Hardesty |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Models
Molecular Circular dichroism Proteases Proteolysis medicine.medical_treatment Analytical chemistry Bacillus Protein Structure Secondary Protein structure Catalytic Domain Electrochemistry medicine General Materials Science Protein Structure Quaternary Protein secondary structure Spectroscopy Protease medicine.diagnostic_test Chemistry Circular Dichroism Hydrolysis Serine Endopeptidases Substrate (chemistry) Surfaces and Interfaces Condensed Matter Physics Ionic strength Biophysics Peptides |
| Zdroj: | Langmuir. 24:13944-13956 |
| ISSN: | 1520-5827 0743-7463 |
| DOI: | 10.1021/la8020386 |
| Popis: | In this work, we studied the interactions of enzymes with model substrate surfaces using label-free techniques. Our model system was based on serine proteases (a class of enzymes that digests proteins) and surface-bound polypeptide substrates. While previous studies have focused on bulk media factors such as pH, ionic strength, and surfactants, this study focuses on the role of the surface-bound substrate itself. In particular, we assess how the substrate density of a polypeptide with an alpha-helical secondary structure influences surface reactivity. An alpha-helical secondary structure was chosen based on literature indicating that stable alpha-helices can resist enzymatic digestion. To investigate the protease resistance of a surface-bound a-helix, we designed an a-helical polypeptide (SS-polypeptide, where SS = disulfide), used it to form films of varying surface coverage and then measured responses of the films to enzymatic exposure. Using quartz-crystal microbalance with dissipation (QCM-D), angle-resolved X-ray photoelectron spectroscopy (AR-XPS), grazing-angle infrared spectroscopy (GAIRS), and other techniques, we characterized the degradation of films to determine how the lateral packing density of the surface-bound SS-polypeptide substrate affected surface proteolysis. Characterization of pure SS-polypeptide films indicated dense packing of helices that maintained their helical structure and were generally oriented normal to the surface. We found that films of pure SS-polypeptide significantly resisted enzymatic digestion, while incorporation of very minor amounts of a diluent in such films resulted in rapid digestion. In part, this may be due to the need for the enzyme to bind several peptides along the peptide substrate within the cleft for digestion to occur. Only SS-polypeptide films that were densely packed and did not permit catalytic access to multiple peptides (e.g., terminal peptides only) were resistant to enzymatic proteolysis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|