Use of biotin derivatives to probe conformational changes in proteins.
Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-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 Lys(826) 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.
Azim-Zadeh
Omid
O
Fachbereich Biologie, Philipps-Universität Marburg, Marburg, Germany.
Hillebrecht
Alexander
A
Linne
Uwe
U
Marahiel
Mohamed A
MA
Klebe
Gerhard
G
Lingelbach
Klaus
K
Nyalwidhe
Julius
J
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry*; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification; D002256:Carbonic Anhydrases / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D008239:Lysine; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D010963:Plasmodium falciparum; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D008565:Membrane Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D001710:Biotin; D002256:Carbonic Anhydrases; D008239:Lysine