Enzymatic proteolysis of a surface-bound alpha-helical polypeptide

Area of Science:

• Biochemistry
• Surface Science
• Enzymology

Background:

• Enzyme activity is influenced by environmental factors like pH and ionic strength.
• Previous research has not fully explored the impact of surface-bound substrates on enzyme-substrate interactions.
• Alpha-helical secondary structures are known for their stability and resistance to enzymatic degradation.

Purpose of the Study:

• To investigate how the density of surface-bound alpha-helical polypeptide substrates affects their interaction with enzymes.
• To understand the role of substrate structure and packing in enzymatic proteolysis at surfaces.
• To explore label-free techniques for studying enzyme-substrate dynamics on surfaces.

Main Methods:

• Utilized quartz-crystal microbalance with dissipation (QCM-D) for real-time monitoring of film degradation.
• Employed angle-resolved X-ray photoelectron spectroscopy (AR-XPS) to analyze surface composition and structure.
• Applied grazing-angle infrared spectroscopy (GAIRS) to characterize the secondary structure of surface-bound polypeptides.
• Designed and synthesized disulfide-crosslinked alpha-helical polypeptides (SS-polypeptides) to form model substrate films.

Main Results:

• Characterization confirmed dense packing and surface-normal orientation of alpha-helices in pure SS-polypeptide films.
• Pure SS-polypeptide films exhibited significant resistance to enzymatic digestion by serine proteases.
• Incorporation of even small amounts of a diluent into the films led to rapid enzymatic digestion.
• Protease resistance was correlated with the inability of the enzyme to access multiple peptide bonds within the enzyme's active site.

Conclusions:

• The lateral packing density of surface-bound alpha-helical substrates critically influences their susceptibility to enzymatic proteolysis.
• Dense packing of alpha-helices, limiting enzyme access to multiple peptide bonds, confers significant protease resistance.
• Surface structure and organization play a crucial role in enzyme-substrate interactions, independent of bulk solution conditions.