Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein

Journal of Peptide Science : an Official Publication of the European Peptide Society
Michael G HoeslNediljko Budisa

Abstract

Aequorea victoria green fluorescent protein and its widely used mutants enhanced green fluorescent protein and enhanced cyan fluorescent protein (ECFP) are ideal target proteins to study protein folding. The spectral signals of their chromophores are directly correlated with the folding status of the surrounding protein matrix. Previous studies revealed that tryptophan at position 57 (Trp57) plays a crucial role for the green fluorescent protein's structural and functional integrity. To precisely dissect its role in ECFP folding, we performed its substitution with the isosteric analogs 4-azatryptophan [(4-Aza)Trp] and 7-azatryptophan [(7-Aza)Trp]. Although Trp is moderately hydrophobic, these isosteric analogs are hydrophilic, which makes them an almost ideal tool to study the role of Trp57 in ECFP folding. We achieved high-level expression of both (4-Aza)Trp-ECFP and (7-Aza)Trp-ECFP. However, great portions (70-90%) of protein samples were insoluble and did not contain a maturated chromophore. All attempts to refold the insoluble protein fractions failed. Nevertheless, low amounts of fully labeled, soluble, chromophore containing fractions with altered spectral features were also isolated and identified. The most probable reas...Continue Reading

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Citations

Nov 29, 2011·Biotechnology Journal·Shun Zheng, Inchan Kwon
Jul 4, 2017·Biotechnology Journal·Vladimir Kubyshkin, Nediljko Budisa
Nov 2, 2013·Nature Communications·Jiun-Yi ShenPi-Tai Chou
Jan 14, 2017·Angewandte Chemie·Federica AgostiniNediljko Budisa
Nov 20, 2015·Journal of the American Association of Nurse Practitioners·Cheryl Toulouse, Marie Kodadek
Mar 16, 2011·Angewandte Chemie·Michael G Hoesl, Nediljko Budisa
Mar 16, 2019·Beilstein Journal of Organic Chemistry·Frank WescheHelge B Bode

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