Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation.
Enzymatic phosphorylation and dephosphorylation reactions were used to modify a genetically engineered variant of spider dragline silk. The approximately 25 kDa protein was phosphorylated with cyclic AMP-dependent kinase and dephosphorylated with calf intestinal alkaline phosphatase. Phosphorylation inhibited beta-sheet assembly of the protein and enhanced solubility to about 5 mg/mL in water, compared to about 20% of this level upon enzymatic dephosphorylation. The cyclability of the phosphorylation-dephosphorylation system was confirmed by MALDI with a model peptide. Kinetic studies conducted with [gamma-(32)P]ATP illustrate that the phosphorylation reaction proceeds over 6 h. Secondary structure of the phosphorylated and dephosphorylated proteins was determined by CD and FTIR. The results illustrate that an enzymatic phosphorylation event can be used to control the solution structure of a protein like silk, which has a tendency to prematurely precipitate due to the formation of beta-sheets.
Winkler
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Department of Chemical and Biological Engineering and Bioengineering Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, USA.
Wilson
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Kaplan
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DL
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D005349:Fibroins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012995:Solubility; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013112:Spiders / Q000201:enzymology* / Q000235:genetics*
D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; C076815:spidroin 2; D005349:Fibroins