Determination of the interface for hTRF2 with telomeric DNA using H/D amid exchange mass spectrometry methods

From the M. Gross Lab: One goal is to develop and apply mass-spectrometry-based methods for foot printing proteins, taking advantage of mass-spectrometry based proteomics analysis. An example is H/D amide exchange to follow conformational changes in proteins, determine their interfaces in binding with other proteins, and quantify the binding affinities of proteins with ligands (PLIMSTEX). Exchange followed by protein digestion, usually with pepsin at low pH (quench conditions) reveals those regions that change upon binding. Examples are the determination of the interface for hTRF2 (human telomeric repeat binding factor) with telomeric DNA (Biochemistry, 47, 1797-1807 (2008)--see graphic) and the identification of the interface of a toxic protein, SPN, from the bacterial pathogen Streptococcus pyogenes with IFS. A complementary foot printing approach is fast photochemical oxidation of proteins (FPOP) whereby OH radicals are laser-generated rapidly in a flow system and react on the microsec time scale with solvent accessible aromatic and sulfur-containing amino-acid residues, thus mapping the protein surface.

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