Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease.
Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis. One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop. The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements. Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates. When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease. Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species. Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein. These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins. The possible mechanisms of protein stabilization are also discussed.
Margarit
I
I
Laboratory of Genetic Engineering and Microbiology, Eniricerche SpA, San Donato Milanese, Milan, Italy.
Campagnoli
S
S
Frigerio
F
F
Grandi
G
G
De Filippis
V
V
Fontana
A
A
D000409:Alanine; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D005998:Glycine; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D010450:Endopeptidases; D000409:Alanine; D005998:Glycine