Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution
Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution
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DOI：
10.1074/jbc.m505303200
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发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Lambolez, B
中科院分区：
文献类型：
--
作者：
Tsuzuki, K;Tricoire, L;Lambolez, B
光蛋白是一种结合钙后发光的光蛋白。通过体外进化结合DNA洗牌和细菌功能筛选，分离出生物发光强度增加或衰减缓慢的Aequorin突变体。在第一轮筛选中分离到发光衰减突变体，其突变位于EF-hand钙结合位点或其附近。在体外进化过程中，突变体群体的发光强度随着有效突变频率的增加而增加，而其他氨基酸取代的频率基本保持稳定。发光强度突变位于His-16或His-169 coelenterazine结合残基附近或位于第一个EF-hand。当在无细胞试验中检查时，没有选择的突变体表现出光子产量的增加。然而，我们观察到两个突变体Q168R和L170I在37℃下表现出光蛋白寿命的增加，这可能是它们在细菌中具有高发光强度的原因。对Q168R和L170I突变的进一步分析表明，它们增加了aequorin的热稳定性。相反，对发光衰减突变体的检测显示，F149S取代降低了aequorin的热稳定性。最后，随机筛选的Gln-168和Leu-170突变体文库证实了这两个位置都参与了热稳定性，并表明通过体外进化选择的Q168R和L170I突变体具有最佳的热稳定性。我们的研究结果表明，Phe-149和Gln-168残基通过将第三个ef手连接到Trp-129和His-169结合残基上，参与了过氧化coelenterazine的稳定和光子发射的触发。
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.