title: Engineering deamidation-susceptible asparagines leads to improved stability to thermal cycling in a lipase.
abstract: At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75°C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90°C. MutC has retained more than 50% activity even after incubation at 100°C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.
authors.affiliation: Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, 500007, India.
authors.forename: K
authors.initials: K
authors.lastname: Bhanuramanand
authors.forename: Shoeb
authors.initials: S
authors.lastname: Ahmad
authors.forename: N M
authors.initials: NM
authors.lastname: Rao
mesh_terms: D000577:Amides / Q000378:metabolism*; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D020134:Catalytic Domain; D004795:Enzyme Stability; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D000577:Amides; D001426:Bacterial Proteins; C079999:LipA protein, Bacteria; D001216:Asparagine
keywords: asparagines; deamidation; lipase; mass spectrometry; site saturation mutagenesis; thermal cycling