Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code

被引：37

作者：

Budisa, N ^{[1
]}

Pal, PP ^{[1
]}

Alefelder, S ^{[1
]}

Birle, P ^{[1
]}

Krywcun, T ^{[1
]}

Rubini, M ^{[1
]}

Wenger, W ^{[1
]}

Bae, JH ^{[1
]}

Steiner, T ^{[1
]}

机构：

[1] Max Planck Inst Biochem, D-82152 Martinsried, Germany

来源：

BIOLOGICAL CHEMISTRY | 2004年 / 385卷 / 02期

关键词：

fluorescence spectroscopy; genetic code; green fluorescent protein structure; non-canonical amino acids; tryptophan;

D O I：

10.1515/BC.2004.038

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The expanded genetic code in combination with sitedirected mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen, chalcogen, and methylcontaining Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange (atomic mutation) and do not affect threedimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to reevaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphuraromatic interactions for avGFP spectral properties and structural stability. The library of novel tailormade avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailormade proteins with interesting and useful spectral properties.

引用

页码：191 / 202

页数：12

共 40 条

[1] Expansion of the genetic code enables design of a novel "gold'' class of green fluorescent proteins [J].