PROTONATION BEHAVIOR OF HISTIDINE-24 AND HISTIDINE-119 IN FORMING THEPH-4 FOLDING INTERMEDIATE OF APOMYOGLOBIN

Heteronuclear NMR methods are used to study the protonation of histidi ne and aspartate residues in the acid-induced unfolding of recombinant sperm whale apomyoglobin. The results are combined with fluorescence and circular dichroism measurements of acid-induced unfolding of wild- type and double mutant (H24V/H119F) proteins. They are consistent with a simple model in which the failure to protonate a single buried hist idine, H24, is largely responsible for the partial unfolding of native (N) wild-type apomyoglobin to the pH 4 folding intermediate (T), H24 is known to form an unusual interaction in which its side chain is bur ied and hydrogen-bonded to the side chain of H119, Two-dimensional H-1 -N-15 heteronuclear NMR spectra indicate that H24 is present in the ra re delta tautomeric farm and remains neutral until N unfolds to I, whi le H119 becomes protonated before the N --> I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N --> I reaction occurs at lower pH. Therefore, the protonation of aspa rtate and/or glutamate residues must provide an additional driving for ce for the N to I reaction, Two-dimensional H-1-C-13 NMR experiments a re used to measure the protonation of aspartates in selectively C-13-l abeled apomyoglobin; the results indicate that none of the aspartate r esidues has a strongly depressed pK(a) in N, as would be expected if i t forms a stabilizing salt bridge.