科研之友
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摘要
The oxygen binding properties of vertebrate hemoglobins (Hb) have been explored in great detail. In contrast, folding and assembly of these heterotetrameric protein complexes remain poorly understood. Similar to investigations of other multisubunit systems, in vitro Hb refolding experiments are often plagued by aggregation. Here we monitor the refolding of bovine Hb by electrospray mass spectrometry (ESI-MS). This technique allows the observation of coexisting subunit combinations, heme binding states, and protein conformers. Exposure to 40% acetonitrile at pH 10 causes Hb disassembly and extensive subunit unfolding. Hb reassembly is triggered by solvent exchange. Experiments conducted at room temperature provide a low meatHb refolding yield. A significantly improved yield is achieved by lowering the temperature to 4 degrees C and by supplementing the protein solution with KCN prior to denaturation. Comparative studies under "low-yield" and "high-yield" conditions report on the interplay between folding and misfolding. The tendency of beta-globin to undergo aggregation is found to be the key impediment to the formation of native Hb. The alpha/beta imbalance generated in this way favors the formation of non-native alpha-globin assemblies. Our data imply that hemin dicyanide formed in the presence of KCN remains weakly bound to denatured beta-globin, thereby counteracting aggregation, such that the refolding yield is enhanced. In the absence of aggregation-related interference, Hb assembly follows a symmetric pathway. Monomeric alpha- and beta-globin adopt a compact conformation upon heme binding. Heme-bound monomers then form heterodimers, and ultimately heterodimer association results in native Hb. This work highlights the utility of time-resolved ESI-MS investigations for interrogating the kinetic competition between on-pathway events and aberrant side reactions during the self-assembly of biomolecular complexes.
- 出版日期2013-3-12
