A vibrational spectral maker for probing the hydrogen-bonding status of protonated Asp and Glu residues

Abstract

Hydrogen bonding is a fundamental element in protein structure and function. Breaking a single hydrogen bond may impair the stability of a protein. We report an infrared vibrational spectral marker for probing the hydrogen-bond number for buried, protonated Asp or Glu residues in proteins. Ab initio computational studies were performed on hydrogen-bonding interactions of a COOH group with a variety of side-chain model compounds of polar and charged amino acids in vacuum using density function theory. For hydrogen-bonding interactions with polar side-chain groups, our results show a strong correlation between the C=O stretching frequency and the hydrogen bond number of a COOH group: approximately 1759-1776 cm(-1) for zero, approximately 1733-1749 cm(-1) for one, and 1703-1710 cm(-1) for two hydrogen bonds. Experimental evidence for this correlation will be discussed. In addition, we show an approximate linear correlation between the C=O stretching frequency and the hydrogen-bond strength. We propose that a two-dimensional infrared spectroscopy, C=O stretching versus O-H stretching, may be employed to identify the specific type of hydrogen-bonding interaction. This vibrational spectral marker for hydrogen-bonding interaction is expected to enhance the power of time-resolved Fourier transform infrared spectroscopy for structural characterization of functionally important intermediates of proteins.

FIGURE 1

The structure of a butyric acid molecule (A) and its hydrogen-bonding interactions with 0, 1, and 2 methanol molecules. The notation, HO–C=O (B), represents that the hydroxyl oxygen atom (in bold and underlined) forms one hydrogen bond with a methanol molecule. Similarly, HO–C=O (C) and HO–C=O (D) indicate that the carbonyl oxygen atom and the hydroxyl hydrogen atom form a hydrogen bond with a methanol molecule, respectively. The notation, HO–C=O (E), denotes that both the hydroxyl hydrogen and the carbonyl oxygen atoms form a hydrogen bond with a methanol molecule.

FIGURE 2

Hydrogen-bond dissociation energy of a protonated carboxylic group (butyric acid) interacting with a methanol molecule. The hydrogen-bond dissociation energies were calculated at selected bond lengths using B3LYP/6-311+G(2d,p) (see Methods for details) for HO–C=O (▵), HO–C=O (□), and HO–C=O (○). The curves are the results of nonlinear least-square fitting of the computational data to Morse potentials. The strongest hydrogen-bonding interaction occurs at 2.94 Å with −11.6 kJ/mol for HO–C=O, 2.87 Å with −18.8 kJ/mol for HO–C=O, and 2.75 Å with −32.1 kJ/mol for HO–C=O.

FIGURE 3

The distributions of calculated and experimental C=O stretching frequencies of a protonated carboxylic group (COOH). (A) The horizontal solid bars show the distributions of calculated C=O stretching frequencies (see Table 4) with no hydrogen-bond interaction (1759–1776 cm⁻¹), one-hydrogen-bond interaction (1733–1749 cm⁻¹), and two-hydrogen-bond interactions (1703–1710 cm⁻¹). The histogram illustrates the distribution of experimental C=O stretching frequencies of 31 buried COOH groups in the steady-state structures of proteins. The horizontal open bars depict the clustered distributions of experimental C=O stretching frequencies that correlate well with computational data. (B) The histograms for experimental C=O stretching frequencies of 31 buried COOH groups in intermediate states (open columns), 31 buried COOH group in the steady states of proteins (solid columns), and the total distributions of both steady states and intermediate states (open and solid columns). The solid and open horizontal bars are the same as those in panel A. The percentage of buried hydrogen bonds were calculated based on experimental data in the frequency regions that mirror the distribution of calculated C=O stretching frequencies with known hydrogen-bond information.

FIGURE 4

A correlation between the calculated hydrogen-bond dissociation energies and the calculated C=O stretching frequencies of a COOH group with neutral and positively charged side-chain groups of amino acids (data shown in Tables 4 and 5). The calculated data are shown in open circles, whereas a linear straight line is the result of nonlinear least-square fitting. The root mean square deviation of the fitting is 7.49 kJ/mol, resulting in with energy in unit of kJ/mol.

FIGURE 5

A 2D frequency distribution of the O–H and C=O stretching modes for probing the specific type of hydrogen-bonding interactions in proteins: HO–C=O (•), HO–C=O (♦), HO–C=O (▪), HO–C=O (□), and HO–C=O (▴). The data are shown in Table 3.