Assembly of Hemoglobin from Denatured Monomeric Subunits: Heme Ligation Effects and Off-Pathway Intermediates Studied by Electrospray Mass Spectrometry
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Bibliographic record
Abstract
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 metHb refolding yield. A significantly improved yield is achieved by lowering the temperature to 4 °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 β-globin to undergo aggregation is found to be the key impediment to the formation of native Hb. The α/β imbalance generated in this way favors the formation of non-native α-globin assemblies. Our data imply that hemin dicyanide formed in the presence of KCN remains weakly bound to denatured β-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 α- and β-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.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it