Measurement of the free and the H-bonded amides of myoglobin

Abstract

It has become abundantly clear that the rather slowly exchanging hydrogens of a protein do not simply relate to α-helix content. Some of the other inferences reached in the early period of protein hydrogen exchange work do still appear to be valid, namely, that most side chain hydrogens exchange “instantaneously” and that H-bonded amides exchange very slowly. However, in conflict with the older equation of slow hydrogens with α-helix are the more recently demonstrated facts that proteins possess H-bonded amides other than those in α-helix, which may also be expected to exchange very slowly, and that amides even when free can exchange measurably slowly, though perhaps not nearly as slowly as those in hydrogen bonds. Thus, rather than absolute separation between “instantaneous” and slow hydrogen exchange classes, differentiation between relatively faster and relatively slower kinetic classes seems indicated. Such a separation might provide a measure of structure representing the over-all number of H-bonded amides rather than just α-helix. A successful test of this expectation was carried out with myoglobin. To perform this test, the fairly rapidly exchanging hydrogens of myoglobin were preferentially labeled by brief exposure to tritiated water, and their exchange-out behavior was studied. The exchange-out of these faster hydrogens convincingly match theoretical exchange curves for free amide groups. These theoretical curves were constructed using the numbers of free amide groups in the myoglobin crystallographic model and hydrogen exchange rates found for such groups in model polymers. By a less demanding variant of this same exchange-in/exchange-out approach, the number of abnormally slowly exchanging hydrogens was independently determined and found to match the number of internally H-bonded amides in the myoglobin model structure. The good agreement found is taken as establishing the identity of the free and the H-bonded amides of myoglobin with distinguishable kinetic classes of exchanging hydrogens. The results demonstrate that, for this protein, free amides show simple and predictable hydrogen exchange behavior, that essentially all H-bonded amides exchange much more slowly, and that essentially all the other side chains exchange considerably faster. Evidence was found for the importance of an “opening”-dependent pathway for slowly exchanging, H-bonded hydrogens of myoglobin, as opposed to some direct exchange mechanism not dependent on conformational movement. The present study also provides another parameter common to both crystalline and dissolved myoglobin, namely, the number of H-bonded (donor) peptide groups.