Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 22.2, pp. 546-547   | 1 | 2 |

Section 22.2.3. Hydrogen-bonding groups

E. N. Bakera*

aSchool of Biological Sciences, University of Auckland, Private Bag 92-109, Auckland, New Zealand
Correspondence e-mail:

22.2.3. Hydrogen-bonding groups

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The hydrogen-bonding capacities of the various hydrogen-bonding groups in proteins are shown in Fig.[link]. All, with the exception of the peptide NH and Trp side-chain NH groups, can participate in more than one hydrogen-bond interaction. Peptide and side-chain C=O groups, for example, can act as acceptors for two hydrogen bonds by using both lone pairs of electrons on the sp2-hybridized oxygen. Likewise, the —OH groups of Ser or Thr can act as donors through their single H atom, and acceptors through their two lone pairs. In Tyr side chains, the C—O bond has some double-bond character, and the phenolic —OH is thus likely to prefer only two hydrogen bonds, both in the ring plane. The carboxylate groups of Asp and Glu are normally ionized above pH 4 and their C—O bonds also have partial double-bond character; each carboxylate oxygen should then be able to accept two hydrogen bonds, although the restriction to two may be less severe than for C=O.


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Hydrogen-bonding potential of protein functional groups. Potential hydrogen bonds are shown with broken lines. Arg, Lys, Asp and Glu side chains are shown in their ionized forms.

Several uncertainties exist. Crystallographically, it is not usually possible to distinguish the amide oxygen and nitrogen atoms of Asn and Gln, and the decision as to which is which has to be made on environmental grounds by considering what hydrogen bonds would be made in each of the two possible arrangements. Likewise, two possibilities exist for His side chains by rotating 180° about Cβ—Cγ. This problem has been analysed by McDonald & Thornton (1994b[link]), and corrections can be made with HBPLUS .

For some side chains, the ionization state is uncertain. Arg and Lys are assumed to be fully protonated, as in Fig.[link], and Asp and Glu are assumed to be fully ionized. Nevertheless, a survey by Flocco & Mowbray (1995[link]) has shown that a small but significant number of short O···O distances between Asp and Glu side chains must represent O—H···O hydrogen bonds, with one carboxyl group protonated. His side chains, in addition to the orientational uncertainty, have a pKa (∼6.5) that implies that they may be in either their neutral or their protonated form, depending on pH and environment. In the neutral form, only one N atom is protonated (more often [\hbox{N}^{\varepsilon 2}], but sometimes [\hbox{N}^{\delta 1}]), but in the protonated form both N atoms carry protons; again, the actual state has to be deduced from their environment. Nucleic acids

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The three components of nucleic acids, i.e. phosphate groups, sugars and bases, all participate in hydrogen bonding to greater or lesser extent. The phosphate oxygen atoms can potentially act as acceptors of two or more hydrogen bonds and are frequently the recipients of hydrogen bonds from protein side chains in protein–DNA complexes. The sugar residues of RNA have a 2′-OH which can act as both hydrogen-bond donor and acceptor, and the 4′-O of both ribose and deoxyribose can potentially accept two hydrogen bonds.

It is the bases of DNA and RNA that have the greatest hydrogen-bonding potential, however, with a variety of hydrogen-bond donor or acceptor sites. Although each of the bases could theoretically occur in several tautomeric forms, only the canonical forms shown in Fig.[link] are actually observed in nucleic acids. This leads to clearly defined hydrogen-bonding patterns which are critical to both base pairing and protein–nucleic acid recognition. The —NH2 and >NH groups act only as hydrogen-bond donors, and C=O only as acceptors, whereas the >N— centres are normally acceptors but at low pH can be protonated and act as hydrogen-bond donors.


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Hydrogen-bonding potential of nucleic acid bases guanine (G), adenine (A), cytosine (C) and thymine (T) in their normal canonical forms.


Flocco, M. M. & Mowbray, S. L. (1995). Strange bedfellows: interactions between acidic side-chains in proteins. J. Mol. Biol. 254, 96–105.Google Scholar
McDonald, I. K. & Thornton, J. M. (1994b). The application of hydrogen bonding analysis in X-ray crystallography to help orientate asparagine, glutamine and histidine side chains. Protein Eng. 8, 217–224.Google Scholar

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