Travelling-wave ion mobility mass spectrometry has been successfully used to probe the gas-phase conformations of the three-dimensional protein structure of the non- covalent complexes of normal and sickle hemoglobin.
Non-tetrameric species were seen which correspond to apo- and holo- forms of theα- andβ-monomers andαhβh-dimers; these are naturally present in solution, and are not products of in-source fragmentation during the ESI process. Both monomer types
have cross-sections similar to each other, suggesting that they maintain a similar folded structure. Extensively disordered monomer structures were not seen, and the similarity between the apo- and holo- forms indicates that the chains are able to retain a folded structure with or without the attachment of a heme group.
A heme-deficient dimer was not observed, and the results suggest that there is no requirement for the association of βa with αh in order for the β-monomer to recruit heme. The data, acquired on fresh blood samples rather than commercially prepared protein, do not support the hypothesis that a heme-deficient dimer is an essential intermediate in the assembly of the hemoglobin tetramer.
The cross-sections calculated for HbA and HbS are comparable to those estimated from published X-ray crystallographic data, supporting other work which investigated the relationship between values obtained from these different techniques. Conformational differences have been observed between the HbA and HbS molecules, a significant structural change caused by a single amino acid substitution. This result, from a relatively well-studied hemoglobin disorder, gives confidence for the use of this method for the investigation of other hemoglobin variants, and also for the study of other metalloproteins.
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