Abstract
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.
Highlights
Namido) hexanoate3 is a hydrophilic reagent that is excluded from most cells and is used for the biotinylation and subsequent affinity purification of cellsurface proteins
Biotinylation of bovine serum albumin (BSA) Is Saturable but Incomplete—SulfoNHS-LC-biotin derivatives possess an active ester group that reacts with the primary amines of proteins and/or the ⑀-amino group of lysine residues, thereby forming an amide bond (Fig. 1)
BSA was biotinylated with increasing molar ratios of sulfo-NHS-LC-biotin to protein
Summary
Biotinylation of BSA and Carbonic Anhydrase—Fatty acidfree BSA (Roth, Karlsruhe, Germany) was dissolved in phosphate-buffered saline (PBS) to a final concentration of 10 mg/ml. The bound peptides were washed with a solution of 0.5% formic acid and eluted from the ZipTipTM columns with 2 l of 33% (v/v) acetonitrile and 0.1% trifluoroacetic acid saturated with ␣-cyano-4-hydroxycinnamic acid directly onto a matrix-assisted laser desorption ionization (MALDI) sample plate and air-dried before analysis in the mass spectrometer. The unbound non-biotinylated peptide fraction was obtained as a supernatant after centrifugation at 10,000 ϫ g for 10 min This fraction was evaporated to dryness and resuspended in 0.1% trifluoroacetic acid before processing for MS. The bound peptides were eluted with 70% acetonitrile, 5% trifluoroacetic acid, and 1 mM D-biotin in 100 mM ammonium bicarbonate at room temperature for 2 h. The latter was computed and visualized using the MOLCAD module of SYBYL 7.1 [19]; hydrogen bond energies were calculated using the MAB force field implemented in MOLOC [20]
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