Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

Abstract

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

Fig. 1.

Some allosterically sensitive hydrogens in Hb measured by tritium exchange and by DXMS (pH 7.4, 0°C); deoxy Hb is blue and Hb liganded by O₂ or CO is red. (A) Hb fully labeled by exchange-in in tritiated water was exchanged-out in oxy and deoxy forms, and with O₂ added back to exchanging deoxy Hb (45). (B) Illustration of functional labeling in an extreme case. Tritium exchange-in was for 1 min at pH 7.4, 0°C (oxy exchange-in/deoxy exchange-out is blue; the reverse process is red). (Inset) The difference curve after subtraction of the background is shown for deoxy Hb (46). (C and D). HX by functional labeling and HPLC fragment separation for the fragments α1–29 and β130–146, respectively. For tritium exchange experiments (filled circles), oxy Hb was initially labeled for 35 min at 0°C, pH 7.4 (24, 32). Triangles show comparative data from DXMS experiments, with oxy Hb initially labeled as just described (open triangles) or more intensively (filled triangles; 20 min, 20°C), which labels additional sensitive sites on α1–29. (HX rates within each set are not monoexponential because different amide hydrogens exchange with somewhat different intrinsic rate constants, k_int in Eq. 2.)

Fig. 2.

MS fragments obtained reproducibly in good yield by using tandem online column-immobilized pepsin and fungal protease XIII.

Fig. 3.

The number of sites measured with the functional labeling protocol used (exchange-in 20°C, 20 min; exchange-out 0°C; upper curve in oxy/out deoxy; lower curve the reverse; pH or pD_read 7.4). Filled circles show exchange-out data measured in whole Hb by established tritium methods. Comparative DXMS results (filled triangles) show the summed deuterium labeling from the fragments in Fig. 4. Equilibrium isotope corrections were applied (29).

Fig. 4.

DXMS results for fragments that span the entire α-chain and β-chain (exchange-in 20 min, 20°C; exchange-out at 0°C, pH or pD_read 7.4). Allosterically sensitive hydrogens in deoxy (blue) and liganded (red) Hb were obtained by subtracting the background from the raw data (both shown in gray). For liganded Hb, samples were initially exchanged out for 1 h in deoxy Hb to lose most of the background label, then switched to the liganded (R state) Hb by dilution into CO-containing buffer. The 1-h background was subtracted. Curves are drawn to fit the deoxy data, then moved onto the time axis to the oxy data to find the rate multiplication factor.