Quantitative measurement of the solvent accessibility of histidine imidazole groups in proteins

We report a method for expressing the solvent accessibility of histidine imidazole groups in proteins. The method is based on measuring the rate of the hydrogen exchange (HX) reaction of the imidazole C(ε1)-hydrogen. The rate profile of the HX reaction as a function of pH gives a sigmoidal curve, which reaches the maximal rate constant (k(max)) on the alkaline side of the sigmoidal curve. To quantitatively describe the solvent accessibility of imidazole groups in proteins, it is necessary to compare the k(max) of the imidazole groups with their intrinsic k(max) ((i)k(max)), the maximal rate constants for the given imidazole groups when they are fully exposed to the bulk solvent. However, the mechanism of the HX reaction suggests that the (i)k(max) of an imidazole group differs depending on its pK(a), and no systematic study has been conducted to clarify how the (i)k(max) is affected by pK(a). We therefore investigated the relationship between (i)k(max) and pK(a) using four imidazole derivatives at three different temperatures. The experimentally determined pK(a)-specific (i)k(max) values allowed us to derive a general formula to estimate the (i)k(max) value of any given imidazole group exhibiting a specific pK(a) at a specific temperature. Using the formula, the protection factors (PF), the ratio of (i)k(max) to k(max), of five imidazole groups in dihydrofolate reductase were obtained and used to express the magnitude of their solvent accessibility. In this definition, the smaller the PF value, the higher the solvent accessibility, and a value of 1 indicates full exposure to the bulk solvent. The solvent accessibility expressed by the PF values agreed well with the solvent accessible surface areas obtained from the X-ray diffraction data.