(A) Crystal structure of E. coli RNase H* (ecRNH*) (PDB: 2RN2) (Katayanagi et al., 1992). Secondary structural elements: Red: Strand 1, Strand 2, Strand 3; Blue: Helix A, Strand 4; Yellow: Helix B, Helix C; Green: Helix D, Strand 5; Purple: Helix E. The core region of the protein (Icore) involving Helix A, Strand 4, Helix B, Helix C, Helix D, Strand 5 and the periphery region of the protein involving Strand 1, Strand 2, Strand 3, Helix E are denoted. (B) Pulsed-labeling setup and workflow. Unfolded, fully deuterated protein in high [urea] is rapidly mixed with low [urea] refolding buffer to initiate refolding. After some refolding time, hydrogen exchange of unprotected amides is initiated by mixing with high-pH pulse buffer. The hydrogen exchange reaction is quenched by mixing with a low-pH quench buffer. The sample is injected onto an LC-MS for in-line proteolysis, desalting, and peptide separation by reverse-phase chromatography followed by MS analysis.