Quantitative Measurement of the Solvent Accessibility of Histidine Imidazole Groups in Proteins

Abstract

We report a method to express the solvent accessibility of histidine imidazole groups in proteins. The method is based on measuring the rate of hydrogen exchange (HX) reaction of the imidazole Cε1-hydrogen. The rate profile of the HX reaction as a function of pH gives a sigmoidal curve, which reaches the maximum rate constant (kmax) 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 kmax of the imidazole groups with their intrinsic kmax (ikmax), the maximum rate constants for the given imidazole groups when they are fully exposed to the bulk solvent. However, the mechanism of HX reaction suggests that the ikmax of an imidazole group differs depending on its pKa, and no systematic study has been conducted to clarify how the ikmax is affected by pKa. We therefore investigated the relationship between ikmax and pKa using four imidazole derivatives at three different temperatures. The experimentally determined pKa-specific ikmax values allowed us to derive a general formula to estimate the ikmax value of any given imidazole group exhibiting a specific pKa at a specific temperature. Using the formula, the protection factors (PF), the ratio of ikmax to kmax, 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 (ASA) obtained from the X-ray diffraction data.

Imidazole C-2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

“…Yet, it is convenient to choose $k_2^0$ for a histidine derivative in which the H/D-exchange reaction is least likely to be interfered with, so that log r for each histidine residue can be estimated graphically on the Brønsted plot as shown in Figure 4. Although the similar linear relationship between $log{k}_{\phi }^{max}$ and p K a for several imidazole derivatives has been suggested to analyze solvent accessibility of histidine residues in a protein, 25 the use of the Brønsted plot drawn on the basis of eq. 11 should allow to compare various histidine residues in different proteins.…”

“…25 However, the exchange rate of His48 was immeasurably slow throughout the pH range tested, despite its location being just below the surface of the protein and thus incompletely shielded from accessing to solvent (Figure S3 in Supporting Information). As a possible factor responsible for the unexpectedly low H/D exchange rate of His48, we note the two hydrogen bonds flanking the imidazole ring (Scheme 2).…”

Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase

“…By contrast, most CCD iron centers are likely capable of binding only a single solvent molecule and have no additional residues with coordination potential in their vicinity (10,44). The presence of anionic Glu side chains in close proximity to the His ligands is expected to elevate the pK a of the inner sphere imidazole rings (64), which in turn would stabilize the His-N ⑀ -iron coordinate bond and tune the reactivity of Fe(II) toward dioxygen. The disrupted interaction between Glu-150 and His-238 in W149A ACO coupled with the severe loss of activity in this mutant emphasize the importance of the 3-Glu sphere.…”

Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen–Deuterium Exchange Mass Spectrometry

“…In this case, when the p K a values are equal, the higher k max values for His 52 and His 80 compared to His 66 , His 69 , and His 84 must be due to differences in the local protein environment, for example, in solvent accessibility or hydrogen bonding patterns. 5,14 …”

“…Because the exchange rate depends on the protonation state of the histidine residue, one can determine histidine p K a ’s by performing this experiment at different pH values. This method has been successfully used to determine the p K a of individual histidine residues in RNase A 4 and dihydrofolate reductase 5 and helped to probe the mechanism by which long-range interactions can stabilize the formation of a complex between anthrax protective antigen and its receptor capillary morphogenesis protein-2. 6 …”