Proton NMR spectroscopic studies of calcium-binding proteins. 2. Histidine microenvironments in .alpha.- and .beta.-parvalbumins as determined by protonation and laser photochemically induced dynamic nuclear polarization effects

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Abstract
The microenvironments of the histidines in three isoforms of Ca(II)-bound parvalbumin (carp, pI = 4.25; pike, pI = 5.00 rat, pI = 5.50) have been examined with 1H NMR techniques to probe their protonation characteristics and photochemically induced dynamic nuclear polarizability (photo-CIDNP). The histidine at position 26 (or 25), present in all three of these proteins, shows absolutely no photo-CIDNP enhancement of its C2H or C5H resonances. Nor does this nonpolarizable histidine possess a normal pKa: values range only from 4.20 for carp to 4.32 for pike to 4.44 for rat. The C2H and C5H resonances of the histidine in this carp isoform split into doublets as the pH is lowered. The magnitude of this splitting depends on the magnetic field strength, temperature, and pH; however, the line intensities within each doublet are temperature-independent. Although the crystal structure of carp paravalbumin indicates that His-26 is exposed to solvent [Kretsinger, R.H., and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313-3326], we conclude that in solution this residue, in its unprotonated state, is part of the hydrophobic core of the protein. In contrast, His-48 in rat parvalbumin and His-106 in pike III parvalbumin shown dramatic photo-CIDNP enhancements of their C2H, C5H, and .beta.-CH2 1H NMR resonances. Combined with its nearly normal pKa, 6.14, and exchange-broadened C2H resonance, the photo-CIDNP enhancement results for His-48 indicate that its microenvironment differs little from random-coil exposure, consistent with its presumed position on the solvent surface of helix C. However, the protonation behavior of His-106 in pike III suggests that its microenvironment is different from random-coil exposure. Not only is its pKa elevated (7.10) and C2H resonance minimally broaded, but its C5H resonance is also extremely sensitive to protonation/deprotonation of His-25. Because of the mutual sensitivity of the C5H resonances of His-25 and His-106 to the protonation/deprotonation of their imidazole rings, we suggest (i) that the protonated form of His-106 is involved in a H bond with a backbone C .dbd. O or side-chain .sbd. OH in the BC-linker region and (ii) that the protonated form of His-25(26) is expelled from the hydrophobic core. Each of these protonations causes a conformational change in the B-helix/F-helix region. In addition, photo-CIDNP studies of the pI = 3.95 isoform of carp parvalbumin indicate that its single potentially enhanceable residue, Tyr-2, is either buried or involved in H-bond formation via its phenolic .sbd. OH group.