A mass spectrometric approach to the study of DNA-binding proteins: interaction of human TRF2 with telomeric DNA.
Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.
Sperry
Justin B
JB
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Shi
Xiangguo
X
Rempel
Don L
DL
Nishimura
Yoshifumi
Y
Akashi
Satoko
S
Gross
Michael L
ML
D000107:Acetylation; D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D016615:Telomere; D035341:Telomeric Repeat Binding Protein 2 / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C504753:TERF2 protein, human; D035341:Telomeric Repeat Binding Protein 2; D004247:DNA