Mass spectrometry and non-covalent protein-ligand complexes: Confirmation of binding sites and changes in tertiary structure

Sharon J. Shields, Olayinka Oyeyemi, Felice C Lightstone, Rod Balhorn

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

An experimental approach is described for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes time resolved limited proteolysis and the high throughput analysis capability of MALDI TOF MS to determine the binding site in a tetanus toxin C-fragment (51 kDa)-doxorubicin (543 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the time resolved limited proteolysis of tetanus toxin C-fragment (TetC) and the TetC-doxorubicin complex every 10 min from 10 to 120 min of digestion revealed that the binding of doxorubicin induced a significant change in surface topology of TetC. Four of the twenty-nine peptides observed by MALDI MS, including amino acids 351-360, 299-304, 305-311 and 312-316, had a lower abundance in the TetC-doxorubicin complex relative to TetC from 10 to 100 min of digestion. A decrease in ion abundance suggests doxorubicin obstructs the access of the protease to one or both termini of these peptides, identifying doxorubicin binding site(s). Conversely, five peptide ions, including amino acids 335-350, 364-375, 364-376, 281-298, and 316-328, all had a greater abundance in the digest of the complex, indicating an increase in accessibility to these sites. These five peptides flank regions of decreased ion abundance, suggesting that doxorubicin not only binds to the surface, but also induces a conformational change in TetC.

Original languageEnglish (US)
Pages (from-to)460-470
Number of pages11
JournalJournal of the American Society for Mass Spectrometry
Volume14
Issue number5
DOIs
StatePublished - May 2003
Externally publishedYes

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Doxorubicin
Mass spectrometry
Mass Spectrometry
Binding Sites
Ligands
Peptides
Proteins
Ions
Proteolysis
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Digestion
Amino Acids
tetanus toxin fragment C
Carrier Proteins
Peptide Hydrolases
Throughput
Topology
Molecules

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

Mass spectrometry and non-covalent protein-ligand complexes : Confirmation of binding sites and changes in tertiary structure. / Shields, Sharon J.; Oyeyemi, Olayinka; Lightstone, Felice C; Balhorn, Rod.

In: Journal of the American Society for Mass Spectrometry, Vol. 14, No. 5, 05.2003, p. 460-470.

Research output: Contribution to journalArticle

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