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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2017: 3.022

CiteScore 2017: 2.81

SCImago Journal Rank (SJR) 2017: 1.562
Source Normalized Impact per Paper (SNIP) 2017: 0.705

Online
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1437-4315
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Volume 386, Issue 11

Issues

Rational engineering of a fluorescein-binding anticalin for improved ligand affinity

Sven Vopel
  • Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Hermine Mühlbach
  • Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Arne Skerra
  • Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2005-11-24 | DOI: https://doi.org/10.1515/BC.2005.126

Abstract

The anticalin FluA is an artificial lipocalin with novelspecificity for the fluorescein group, which was engineered from an insect bilin-binding protein by targeted random mutagenesis and selection. Based on the crystal structure of FluA, an attempt was made to improve the complementarity of its ligand pocket to fluorescein by rational protein design. Several side chains participating in sub-optimal interactions with the ligand were identified and replaced by residues that promised a better steric fit. As a result, the substitution of Ala45 by Ile and of Ser114 by Thr or Arg led to a tight affinity of ca. 1 nM, which is approximately 30-fold better than that of the parental anticalin. Similar to the original FluA, the improved version shows almost complete quenching of the bound ligand fluorescence. Interestingly, the quenching effect was significantly reduced when Trp129 was replaced by Tyr, thus supporting the previously postulated role of this residue, which closely packs against the bound ligand, for efficient electron transfer to the excited fluorescein. Circular dichroism spectra revealed that all variants investigated had retained the lipocalin fold. Corresponding thermal unfolding experiments confirmed similar folding stabilities, with melting temperatures ranging from 52.9 to 60.5°C (i.e., for the high-affinity variant).

Keywords: circular dichroism; fluorescence titration; hapten; ligand binding; lipocalin; protein design; spectroscopy

About the article

Corresponding author


Received: May 15, 2005

Accepted: August 8, 2005

Published Online: 2005-11-24

Published in Print: 2005-11-01


Citation Information: Biological Chemistry, Volume 386, Issue 11, Pages 1097–1104, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2005.126.

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