Biosynthetic incorporation of 7-azatryptophan into the phage lambda lysozyme: estimation of tryptophan accessibility, effect on enzymatic activity and protein stability.
The phage lambda lysozyme (lambda L) contains four tryptophans. These have been efficiently replaced by 7-azatryptophan (7aW) through biosynthetic incorporation into the overexpressed protein. Comparative analysis of the effect of temperature or pH on the fluorescence of the wild-type lambda L and 7aWs-containing protein (a lambda L) shows that the stability of the protein is only mildly reduced by 7aW incorporation above pH 5 but that it is strongly decreased below pH 4 on protonation of inaccessible 7aWs. The a lambda L fluorescence depends on pH as a consequence of its effect on the denaturation equilibrium, on the state of protonation of accessible 7aWs in the native state and of all 7aWs in the denatured state. The pH dependence of the fluorescence is used to estimate the number of accessible tryptophans in the protein. The result agrees with that derived from tryptophan NH exchange measurements by 1H-NMR. The acid limb of the activity-pH profile is characterized by a sharp drop that might arise from a cooperative acid-induced denaturation. The difference in acid stability of a lambda L versus lambda L is used to rule out this acid denaturation hypothesis as tryptophan replacement does not affect the lytic activity on chloroform-sensitized Escherichia coli cells or its pH profile.
Soumillion
P
P
Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Belgium.
Jespers
L
L
Vervoort
J
J
Fastrez
J
J
D010582:Bacteriophage lambda / Q000201:enzymology*; D004795:Enzyme Stability; D005453:Fluorescence; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
C006657:7-azatryptophan; D014364:Tryptophan; D009113:Muramidase