Thermal stability determinants of chicken egg‐white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules

Abstract

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 5 5 , and 91. The midpoint temperature of thermal denaturation transition (T,) values of these core constructs range from 60.9 to 77.3 "C, extending an earlier, more limited investigation on thermostability. The T,,, values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9A) and 155L/S91T/DlOlS (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of T, for the S91D mutant was compared with that of WT enzyme. The pK, of S91D is 1.2 units higher in the native than in the denatured state, corresponding to AAG298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.

An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device

“…The melting temperature of lysozyme (74 ± 0.5 °C) w as previously determined using different methodology (18–21). A comparison between Sypro-orange and BFC was made with a lysozyme concentration at 2.25 mg/ml (Fig.…”

The “Two-state Folder” MerP Forms Partially Unfolded Structures that Show Temperature Dependent Hydrogen Exchange

“…7,16 The change in free energy for protein unfolding as a function of temperature is described by the Gibbs -Helmholtz equation: 16,17 …”

Mutagenesis-based definitions and probes of residue burial in proteins

“…Charged residue substitutions at these sites were always found to inactivate the protein. Six additional examples of effects of Asp substitutions of buried, noncharged residues were obtained from the Protherm database (http:͞͞gibk26.bse.kyutech.ac.jp͞jouhou͞ protherm͞protherm search.html): A52D and I53D in RNaseH (36,37), L133D in case of T4 lysozyme (38), S91D in hen egg white lysozyme (39), Y282D in case of Lac repressor (40), and Y78D in U1A protein (41). In all of the cases, there was a drastic decrease in protein thermodynamic stability relative to WT.…”