Document Type
Article
Publication Title
Protein Science
Abstract
A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 °C and no activity at 43 °C has been selected. These mutants, designated 'tight' ts mutants, differ from most other T4 lysozyme mutants that are active at 43 °C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T1571, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 °C. Two mutants were identified: L91P and L66P. Both insert proline residues into α-helical regions of the WT protein structure. The stabilities (ΔΔG) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40-ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the α-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain α-helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent-filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge.
First Page
742
Last Page
751
DOI
10.1002/pro.5560050419
Publication Date
1-1-1996
Recommended Citation
Gray, Terry M.; Arnoys, Eric J.; Blankespoor, Stephen; and Born, Tim, "Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: Leucine 66 to proline and leucine 91 to proline" (1996). University Faculty Publications and Creative Works. 485.
https://digitalcommons.calvin.edu/calvin_facultypubs/485