Abstract
Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.
Highlights
Proteins are made up of different amino acids that carry unique properties and responsible for folding, function, stability, and solubility
The staphylococcal lipase sequences were aligned with other homologous sequences from the genus Bacillus and Pseudomonas and to our expectation, distribution of lysine in Bacillus and Pseudomonas lipases is random and not conserved (Figure S1)
The structural and dynamics study of the WT and mutant lipases provide insights on the structural properties and behaviour of the protein conformation when simulated in water environment
Summary
Proteins are made up of different amino acids that carry unique properties and responsible for folding, function, stability, and solubility. Protein stability is a crucial property as it determines protein functionality. It is well accepted that factors, such as hydrogen bonds, hydrophobic effect, ion pairs, and salt bridges are essential to promote protein stability. Apart from the intramolecular interaction, surface charge has been considerably recognized as one of the important aspects that can alter protein properties [1,2]. In a typical globular protein, one third of the amino acids on the protein-water interface are charged residues [3]. Five amino acid residues (Glutamate, Aspartate, Arginine, Lysine, and Histidine)
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