Effects of removal of the N-terminal amino acid residues on the activity and conformation of firefly luciferase.
A series of deletion mutants were constructed using polymerase chain reaction (PCR) to investigate the roles of luciferase N-terminal residues. The coding sequences of the first 0 (Luc0), 6 (Luc6), 7 (Luc7), 8 (Luc8), 9 (Luc9), 10 (Luc10) and 20 (Luc20) amino acids of the N-terminus were deleted and inserted into the prokaryotic expression vector pBV220. The results showed that the enzymes were completely inactivated when the first eight or more N-terminal amino acids were removed. The recombinant Luc0 and mutants Luc6 and Luc7 were purified to homogeneity by ammonium sulfate precipitation and liquid chromatography for determination of their activity and conformational changes. The activity assay showed that removal of the first six amino acids resulted in 29% loss of enzymatic activity while removal of the first seven amino acids resulted in nearly complete inactivation (with remaining activity <0.5% of the original activity). Circular dichroism spectra showed no significant secondary structure changes. But the fluorescence emission maximum red-shift indicated some conformational changes. Luc6 and Luc7 were more sensitive to guanidine unfolding than Luc0. The present result indicated the significant role of Ile7 to the luciferase stability.
Wang
Xi Cheng
XC
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, PR China.
Yang
Jian
J
Huang
Wei
W
He
Lin
L
Yu
Jiang Tao
JT
Lin
Qing Song
QS
Li
Wei
W
Zhou
Hai Meng
HM
D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D008156:Luciferases; D007930:Leucine