Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline.
A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 degrees C and no activity at 43 degrees C has been selected. These mutants, designated "tight" ts mutants, differ from most other T4 lysozyme mutants that are active at 43 degrees 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 degrees C. Two mutants were identified: L91P and L66P. Both insert proline residues into alpha-helical regions of the WT protein structure. The stabilities (delta delta G) 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 alpha-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain alpha-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.
Gray
T M
TM
Department of Chemistry and Biochemistry, Calvin College, Grand Rapids, Michigan 49546, USA. grayt@calvin.edu
Arnoys
E J
EJ
Blankespoor
S
S
Born
T
T
Jagar
R
R
Everman
R
R
Plowman
D
D
Stair
A
A
Zhang
D
D
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D007930:Leucine / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000494:pharmacology
D014508:Urea; D011392:Proline; D009113:Muramidase; D007930:Leucine