Amide H,D Exchange

Significantly reduced rates for proton-deuterium exchange prove that the corresponding amide proton is either involved in stable hydrogen bonds, shielded from solvent access or both. Because of the ambiguity in interpretation, additional information about the persistence of hydrogen bonds stemming from structure calculations or from relaxation data should be available. 

NMR techniques are unique in their ability to resolve internal dynamics with site-specific probes. Backbone dynamics may be derived from relaxation data of 15N nuclei. Relaxation data are conveniently measured in experiments that utilize [15N,1H]-HSQC-derived pulse sequences and hence can be performed within less than a week of total instrument time with a 1 mM sample (at one field). The underlying principle of the measurement is described in Chapt. 12 and has also been recently reviewed by Palmer [91]. 

Whereas 15N longitudinal and transverse relaxation rates can be determined with sufficient precision, the determined values of the 15N 1H -NOE differ significantly from the true values for residues involved in fast amide proton exchange. A comparison between the values for the 15N 1H -NOE for NPY in solution in the presence and in the absence of DPC is displayed in the Fig. 5.9. The comparison reveals two striking differences. 

Firstly, the (negative) values of the NOE for residues of the unstructured N-terminus that do not interact with the DPC micelle surface are larger. This result is most probably due to increased saturation transfer from the water and results from increased exchange of amide protons at the used pH of 6.0 compared to that used in the absence of DPC (pH 3.1). Secondly, the values for residues from the C-terminal pentapeptide are negative in the case of NPY free in solution whereas they are positive in the micelle-bound form. This clearly indicates that the C-terminal pentapeptide is significantly rigidified upon binding to the micelle. The result is supported by the structure calculation that displays rather low RMSD values for that part 

NPY free in solution (black bars) and NPY bound micelles, to micelles (grey bars) (top), and (bottom) gener- 

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NMR of Membrane-Associated Peptides and Proteins 5.3.3.2 Amide H,D Exchange... 

M. M. Zhu, D. L. Rempel, M. L. Gross Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein—ligand binding constants. J. Am. Soc. Mass Spectrom. 2004, 15, 388-397. 

M.L. Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants. J. Am. 

Henzler-Wildman, K., Kern, D. (2007) Dynamic personalities of proteins. Nature, 450 (7172), 964-972. Keppel, T.R., Howard, B.A., Weis, D.D. (2011) Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry. Biochemistry, 50 (40), 8722-8732. 

Moreover, the kinetics of amide H/D exchange can be measured also by NMR and MS spectroscopy this latter has been emerged as an attractive alternative to NMR method, with significant contributions to the imderstanding of the role of protein dynamics in eri2yme-catalyzed reactions (Kaltashov Eyles, 2002). 

Fig. 5. Comparison of ab initio, DFT/BPW91/6-31G -computed IR and VCD spectra over the amide I, II, and III regions for model peptides (of the generic sequence Ac-Alaw-NHCH3). These are designed to reproduce the major structural features of an o -helix (top left, n— 6, in which the center residue is fully H-bonded), a 3i helix (PLP Il-like, top right, n— 4), and an antiparallel /1-sheet (n= 2, 3 strands, central residue fully H-bonded) in planar (bottom left) and twisted (bottom right) conformations. The computations also encompass all the other vibrations in these molecules, but those from the CH3 side chains were shifted by H/D exchange (CH3) to reduce interference with the amide modes.

Fig. 10. Comparison of VCD spectra of four proteins in H2O (left, amide I + II) and D2O (right, amide V + IF) with dominant secondary structure contributions from G -helix (myoglobin, MYO, top), /3-sheet (immunoglobin, IMUN), both helix and sheet (lactoferrin, LCF) and no structure (o -casein, CAS, bottom). The comparisons emphasize the distinct band shapes developed in the amide I and V for each structural type. Note the reduced S/N in the F O-based spectra and the shape changes upon H/D exchange for helix and sheet (and mixed) structures, but relatively little for the unstructured CAS.

Table 11.2 Average rate constants and number of amide hydrogens in each rate group for H/D exchange of ras-CDP under different Mg concentrations. Measurements were made in 100 mM KCI/50 mM HEPES, 90% D2O, apparent pH of 7.4, T = 21.5 + 0.5 °C. The final [ras-GDP]tot in H/D exchange media was 1.5 pM. EDTA was used to control [Mg +] in solution.

To initiate an H/D exchange reaction, a protein sample, initially in non-deuterated buffer, is incubated in a buffer with 50-90% mole fraction deuterated water. There are almost no restrictions on reaction conditions which allow exchange behavior to be studied as a function of protein and buffer composition, solution pH, and in the presence and absence of ligands. To follow the deuterium buildup of individual amide hydrogen or sets of hydrogens, several on exchange time points are sampled for each condition. 

Hamuro Y, Zawadzki K.M., Kim J.S., Stranz D., Taylor S.S., Woods V.L. Jr Dynamics of cAPK type 11b activation revealed by enhanced amide H/2H exchange mass spectrometry (DXMS). J. Mol. Biol. 2003, 327, 1065-1076. 

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