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J. Biol. Chem., Vol. 282, Issue 30, 21609-21617, July 27, 2007

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Use of Biotin Derivatives to Probe Conformational Changes in Proteins^*

Omid Azim-Zadeh¹, Alexander Hillebrecht, Uwe Linne^¶, Mohamed A. Marahiel^¶, Gerhard Klebe, Klaus Lingelbach², and Julius Nyalwidhe

From the Fachbereich Biologie, the Institut für Pharmazeutische Chemie, and the ^¶Fachbereich Chemie/Biochemie, Philipps-Universitaät Marburg, D-35032 Marburg, Germany

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the -amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys⁸²⁶ in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83–87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Received for publication, November 27, 2006 , and in revised form, May 16, 2007.

^* This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 593 (to K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Tables 1–3.

¹ Recipient of a scholarship from the German Academic Exchange Service.

² To whom correspondence should be addressed: Fachbereich Biologie, Philipps-Universitaät Marburg, Karl von Frisch Str. 8, D-35032 Marburg, Germany. Tel.: 49-6421-282-3404; Fax: 49-6421-282-1531; E-mail: lingelba{at}Staff.Uni-Marburg.de.

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