Determination of the rotational dynamics and pH dependence of the hydrogen exchange rates of the arginine guanidino group using NMR spectroscopy

Summary

The dynamic behaviour of the guanidino group of arginine has been investigated quantitatively with the intention of providing a set of basis values for the interpretation of data acquired for arginine residues in proteins. At room temperature, a single broad resonance line is observed for the four η-NH₂ protons. Upon cooling the sample (≈10°C at 500 MHz), two η-NH₂ proton resonances are resolved which were shown by HMQC spectroscopy to be the result of slowed rotation about the N_ε−C_ζ partial double bond. The flip rate (k_NC) about the N_ε−C_ζ bond was measured as a function of temperature using line-shape analysis of both ¹H and ¹⁵N NMR spectra; at 25°C, k_NC is between 900 and 1000 s^-1. The exchange broadening, due to N_ε−C_ζ bond flips, typically results in weak or missing signals for the η-NH₂ protons of arginine residues in HMQC or INEPT experiments recorded at room temperature, unless the motion is restricted in some way. In a related series of experiments, the pH dependence of the hydrogen exchange rates of the ε-NH and η-NH₂ protons of arginine was measured using saturation transfer ¹H NMR spectroscopy and compared with the equivalent NH₂ protons of the guanidinium ion. As expected, OH ion catalysis dominates over most of the pH range and proceeds at a rate close to the diffusion limit for both types of proton (k_OH=2×10⁹−1×10¹⁰ M^-1s^-1, depending on conditions). At low pH values, however, catalysis by H₃O⁺ becomes important and leads to characteristic rate minima in the exchange versus pH profiles. Acid catalysis is significantly more effective for the η-NH₂ protons than for the ε-NH proton; at low ionic strength (50 mM KCl) the rate minima occurred at pH 3.6 and 2.3, respectively. Under these conditions, acid-catalysed rate constants (k_H) of 706 M^-1s^-1 (η-NH₂) and 3 M^-1s^-1 (ε-NH) were obtained at 25°C. At high ionic strength (1 M KCl) the rate of OH^- ion catalysis is decreased slightly, whereas the H₃O⁺-catalysed rate is unchanged. The k_OH value of the free guanidinium ion is identical to that of the η-NH₂ protons but acid catalysis occurs less easily, leading to a rate minimum at pH 3.3.