Strength of a bifurcated H bond

Abstract

Macromolecules are characterized by their particular arrangement of H bonds. Many of these interactions involve a single donor and acceptor pair, such as the regular H-bonding pattern between carbonyl oxygens and amide H(+)s four residues apart in α-helices. The H-bonding potential of some acceptors, however, leads to the phenomenon of overcoordination between two donors and one acceptor. Herein, using isotope-edited Fourier transform infrared measurements and density functional theory (DFT) calculations, we measured the strength of such bifurcated H bonds in a transmembrane α-helix. Frequency shifts of the (13)C=(18)O amide I mode were used as a reporter of the strength of the bifurcated H bond from a thiol and hydroxyl H(+) at residue i + 4. DFT calculations yielded very similar frequency shifts and an energy of -2.6 and -3.4 kcal/mol for the thiol and hydroxyl bifurcated H bonds, respectively. The strength of the intrahelical bifurcated H bond is consistent with its prevalence in hydrophobic environments and is shown to significantly impact side-chain rotamer distribution.

Keywords: FTIR; membrane proteins; protein structure.

Fig. 1.

(A) Sequence of the M2 peptide used in the analysis. Residues in black or red were isotopically labeled with 1-C=O. Val27 is in red because it is the only residue in the sequence that has a hydroxylic residue four residues in its C-terminal direction, as indicated by the dotted arrow. Note that numbering is according to the sequence of the full length protein. (B) FTIR spectra in the region of the isotope-edited amide I peak of 10 M2 transmembrane peptides in hydrated lipid bilayers (35). The different peptides are labeled with a 1-C=O at the position indicated on the ordinate. The shaded region represents the peak center range for all peptides except for the peptide labeled at Val27. The wavenumber shift (cm) between the Val27 peak and the average of all of the other peptides is indicated.

Fig. 2.

(A) Schematic structures of the different H-bonding configurations in the four different peptides in the region of residue number Phe26 and 30 that were used for FTIR experimental measurements. The isotopically (C and O) labeled carbonyl group of Phe26 is depicted in red. The main-chain (canonical) and bifurcated H bonds are shown in orange and purple, respectively. (B) FTIR spectra in the region of the isotope-edited amide I peak of four SARS E peptides in hydrated lipid bilayers. The different peptides are: Val30 (black), Cys30 (brown), Thr30 (green), and Ser30 (cyan). The wavenumber shifts relative to the peptide with a valine at position 30 are indicated.

Fig. 3.

Structures of the systems used in the DFT calculations. All calculations contained N-Methylacetamide as the H-bond acceptor (top molecule) with different potential H-bond donors as the bottom molecule: (A) N-Isobutylacetamide (valine mimic), (B) N-(2-Hydroxyethyl)acetamide (serine mimic), and (C) N-(2-Thioethyl)acetamide (cysteine mimic). Systems D and E are equivalent to B and C, respectively, but in rotameric configuration (+gauche) that does not facilitate bifurcated H bonding (angle rotation in green). The N-Methylacetamide backbone in the donors is shown in gray. The main-chain (canonical) and bifurcated H bonds are shown in orange and purple, respectively. The isotopically ( C and O) labeled carbonyl group of N-Methylacetamide is depicted in red.

Fig. 4.

Distribution of serine rotamers as a function of side-chain exposure in (Upper) helices and (Lower) nonhelical elements. The data set was a nonhomologous representation of all solved water-soluble proteins (37). Exposure was calculated as a ratio between the exposure of the specific serine and the maximum exposure.