The combination of hydrogen/deuterium exchange or chemical cross-linking techniques with mass spectrometry: mapping of human 14-3-3ζ homodimer interface

Journal of Structural Biology
Kateřina HaladováMiroslav Sulc

Abstract

Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have bee...Continue Reading

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Citations

Dec 19, 2012·Biochimica Et Biophysica Acta·A KonijnenbergF Sobott
Jun 19, 2014·Proceedings of the National Academy of Sciences of the United States of America·Alexander LeitnerRuedi Aebersold
May 6, 2015·Methods : a Companion to Methods in Enzymology·Roland F Rivera-SantiagoDavid W Speicher
May 9, 2015·Mass Spectrometry Reviews·Jinyu LiRita Grandori
Nov 20, 2012·FEBS Letters·Nikolai N Sluchanko, Nikolai B Gusev
Nov 15, 2014·Analytical Chemistry·Gregory F PirroneJohn R Engen

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