Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive.
A number of mutations have been shown previously to stabilize T4 lysozyme. By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (delta delta G = 3.6 kcal/mol). This shows that it is possible to engineer a protein of enhanced thermostability by combining a series of rationally designed point mutations. It is also shown that this stabilization is achieved with only minor, localized changes in the structure of the protein. This is consistent with the observation that the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associated with the constituent single mutants. One of the seven substitutions, Asn116-->Asp, changes a residue that participates in substrate binding; not surprisingly, it causes a significant loss in activity. Ignoring this mutation, there is a gradual reduction in activity as successively more mutations are combined.
Zhang
X J
XJ
Howard Hughes Medical Institute, University of Oregon, Eugene 97403, USA.
Baase
W A
WA
Shoichet
B K
BK
Wilson
K P
KP
Matthews
B W
BW
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering; D013816:Thermodynamics
D009113:Muramidase