Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution.
Aequorin is a photoprotein that emits light upon binding calcium. Aequorin mutants showing increased intensity or slow decay of bioluminescence were isolated by in vitro evolution combining DNA shuffling and functional screening in bacteria. Luminescence decay mutants were isolated at the first round of screening and carried mutations located in EF-hand calcium binding sites or their vicinity. During in vitro evolution, the luminescence intensity of the population of mutants increased with the frequency of effective mutations whereas the frequency of other amino acid substitutions remained roughly stable. Luminescence intensity mutations neighbored the His-16 or His-169 coelenterazine binding residues or were located in the first EF-hand. None of the selected mutants exhibited an increase in photon yield when examined in a cell-free assay. However, we observed that two mutants, Q168R and L170I, exhibited an increase of the photoprotein lifetime at 37 degrees C that may underlie their high luminescence intensity in bacteria. Further analysis of Q168R and L170I mutations showed that they increased aequorin thermostability. Conversely, examination of luminescence decay mutants revealed that the F149S substitution decreased aequorin thermostability. Finally, screening of a library of random Gln-168 and Leu-170 mutants confirmed the involvement of both positions in thermostability and indicated that optimal thermostability was conferred by Q168R and L170I mutations selected through in vitro evolution. Our results suggest that Phe-149 and Gln-168 residues participate in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues.
Tsuzuki
Keisuke
K
Laboratoire de Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, Ecole Supérieure de Physique et de Chimie Industrielles, 75005 Paris, France.
Tricoire
Ludovic
L
Courjean
Olivier
O
Gibelin
Nathalie
N
Rossier
Jean
J
Lambolez
Bertrand
B
D000331:Aequorin; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000032:analysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D049449:Luminescence; D008969:Molecular Sequence Data; D017785:Photons; D013696:Temperature
D004269:DNA, Bacterial; D000331:Aequorin