The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family

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.

Antibacterial efficacy of in‐house designed cell‐penetrating peptide against multi‐drug resistant strains of Salmonella Enteritidis and Salmonella Typhimurium

“…Furthermore, on subjecting the P7 peptide at 90°C, a twofold increase in the MIC, as well as MBC values, was observed at 15‐ and 30‐min time intervals respectively (Supplementary Tables 2a, 2b). Although the arginine residues of P7 and APP were associated with thermal stability (Ahmad et al ., 2020), their interactions with other residues might have adversely impacted the thermostability (Shah et al ., 2011).…”

Effect of multi arm-PEG-NHS (polyethylene glycol n-hydroxysuccinimide) branching on cell adhesion to modified decellularized bovine and porcine pericardium

“…50 Ninhydrin also reacts with the free amine of a lysine residue on the surface of the tissue. 51–53 The ninhydrin assay demonstrated that modification with a 1 : 1 ratio provided suitable conditions for the reaction of the PEG molecules and the NH 2 groups of the pericardium for all multi-arm PEGs. Increasing the modification ratio to 1 : 2 achieved stable surface modification.…”