Identification of biotinylation sites on proteins by selective retrieval of 2-iminobiotinylated peptides from proteolytic peptide mixtures: localization of the accessible lysine residues on the photosystem I subunits PsaD and PsaE.
An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains. Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively. 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography. The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD. No unmodified peptides of PsaD or PsaE were detected. According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus. Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible.
von Leoprechting
A
A
Biologisches Institut II, Universität Freiburg, Schänzlestrasse 1, Freiburg, D-79104, Germany.
Hörth
P
P
Haehnel
W
W
Schilz
E
E
Mühlenhoff
U
U
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000032:analysis; D019921:Biotinylation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000737:chemistry; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism
D000345:Affinity Labels; D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; D011506:Proteins; C070290:PsaD protein, cyanobacteria; D011994:Recombinant Proteins; C066402:avidin-agarose; C079741:photosystem I, psaE subunit; D001360:Avidin; D001710:Biotin; D012685:Sepharose; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; C028072:2-iminobiotin; D008239:Lysine