Measurement of histidine pKₐ values and tautomer populations in invisible protein states
2014
Hansen, Alexandar L. | Kay, Lewis E.
The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK ₐs and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often “invisible” in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine ¹³C ᵧ, ¹³C δ₂ chemical shifts and ¹H ε- ¹³C ε one-bond scalar couplings. The methodology is first validated and then used to obtain pK ₐ values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pK ₐ values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5–6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.
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