Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation

Area of Science:

• Biochemistry
• Materials Science
• Protein Engineering

Background:

• Spider dragline silk is a protein with a tendency to aggregate.
• Controlling silk's solubility is crucial for its application.
• Enzymatic modification offers a potential method for silk protein engineering.

Purpose of the Study:

• To investigate the use of enzymatic phosphorylation and dephosphorylation to control the solution structure and solubility of a genetically engineered spider silk variant.
• To determine the impact of these modifications on the silk's secondary structure and assembly properties.

Main Methods:

• Genetically engineered spider silk variant (approx. 25 kDa) was subjected to enzymatic phosphorylation using cyclic AMP-dependent kinase.
• Dephosphorylation was performed using calf intestinal alkaline phosphatase.
• Protein solubility and secondary structure were analyzed using techniques including circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR).
• Mass spectrometry (MALDI) was used to confirm the cyclability of the modification system.

Main Results:

• Phosphorylation significantly inhibited beta-sheet assembly, leading to enhanced protein solubility (approx. 5 mg/mL in water).
• Dephosphorylation reversed this effect, reducing solubility.
• Kinetic studies showed phosphorylation proceeded over 6 hours.
• CD and FTIR analyses confirmed changes in secondary structure upon phosphorylation and dephosphorylation.

Conclusions:

• Enzymatic phosphorylation is an effective method to regulate the solution structure of spider silk proteins.
• This enzymatic control can prevent premature precipitation and enhance silk solubility for potential applications.
• The phosphorylation-dephosphorylation system demonstrates cyclability, offering a tunable approach to silk protein processing.