Hydrogen atoms in proteins: positions and dynamics

Abstract

Hydrogen atoms constitute about half of the atoms in proteins. Thus they contribute to the complex energy landscape of proteins [Frauenfelder, H., Sligar, S. G. & Wolynes, P. G. (1991) Science 254, 1598-1603]. Neutron crystal structure analysis was used to study the positions and mean-square displacements of hydrogen in myoglobin. A test of the reliability of calculated hydrogen atom coordinates by a comparison with our experimental results has been carried out. The result shows that >70% of the coordinates for hydrogen atoms that have a degree of freedom is predicted worse than 0.2 A. It is shown that the mean-square displacements of the hydrogen atoms obtained from the Debye-Waller factor can be divided into three classes. A comparison with the dynamic mean-square displacements calculated from the elastic intensities obtained from incoherent neutron scattering [Doster, W., Cusack, S. & Petry, W. (1989) Nature 337, 754-756] shows that mainly the side-chain hydrogen atoms contribute to dynamic displacements on a time scale faster than 100 ps.

Fig. 1.

(a and b)2F_exp - F_calc scattering densities of Leu-32 (a) and hydrogen bridge (arrow) between Pro-100 and Tyr-103 (b) [dark gray, positive density (1.5σ); light gray, negative density (-1.5σ)]. (c and d) Δρ maps. (c) Density disappears at light gray and reappears at dark gray contours. (d) Density disappears at dark gray and reappears at light gray contours. Pictures were generated with the program O (35).

Fig. 2.

Comparison of experimentally determined and calculated hydrogen positions. Shown are Δρ maps (green, positive; red, negative). (a) Only differences for ¹H atoms. (b) Only differences for ²H atoms. Pictures were generated with the program O (35).

Fig. 3.

Histogram of the rms differences (rmsd) between the coordinates of experimentally determined and predicted hydrogen atoms for hydrogen atoms in methyl groups (a), and for the exchangeable backbone hydrogen atoms. (b) Filled histogram, weak planarity restraint; unfilled histogram (first column), standard planarity restraint.

Fig. 4.

Mean-square displacements of hydrogen atoms (only ¹H). (a) All atoms (open bars), bonded to main-chain atoms (hatched bars), and hydrogen atoms in side chains (filled bars). (b) All hydrogen atoms in side chains (open bars), hydrogen atoms in methyl groups (filled bars), and lysine groups (hatched bars). (c) Least-squares fit (solid line) of the histogram for all hydrogen atoms (diamonds) with three Gaussian distributions (dashed lines).

Fig. 5.

(a) Fit of the elastic intensity from incoherent neutron scattering with the model of three different classes of hydrogen atoms. Data are taken from Doster et al. (25). ▵ (largest intensity values), 202 K; ○, 242 K; □, 277 K; ▿, 320 K. (Inset) Dashed lines, contributions of the three classes at 320 K. (b) Open symbols, temperature dependence of the dynamic mean-square displacement determined by the fits as shown in a. ▵, main-chain-like; □, methyl-like; ⋄, lysine-like; filled symbols, the displacements determined from the structure analysis. (Inset) ○, mean-square displacements taken from ref. ; solid line, harmonic part of the mean-square displacements taken from ref. ; ▪, average mean-square displacements from Eq. 3.