Partial acetylation of lysine residues improves intraprotein cross-linking.
Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.
Guo
Xin
X
Department of Pharmaceutics and Medicinal Chemistry, University of the Pacific, Stockton, CA 95211, USA.
Bandyopadhyay
Pradipta
P
Schilling
Birgit
B
Young
Malin M
MM
Fujii
Naoaki
N
Aynechi
Tiba
T
Guy
R Kiplin
RK
Kuntz
Irwin D
ID
Gibson
Bradford W
BW
D000085:Acetates / Q000737:chemistry; D000107:Acetylation; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
D000085:Acetates; D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D012997:Solvents; D013388:Succinimides; C088219:sulfosuccinimidyl acetate; D045304:Cytochromes c; D012259:Ribonuclease, Pancreatic; D008239:Lysine