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Submillisecond protein folding events monitored by rapid mixing and mass spectrometry-based oxidative labeling.
- Source :
-
Analytical chemistry [Anal Chem] 2013 Sep 17; Vol. 85 (18), pp. 8618-25. Date of Electronic Publication: 2013 Jul 26. - Publication Year :
- 2013
-
Abstract
- Kinetic measurements can provide insights into protein folding mechanisms. However, the initial (submillisecond) stages of folding still represent a formidable analytical challenge. A number of ultrarapid triggering techniques have been available for some time, but coupling of these techniques with detection methods that are capable of providing detailed structural information has proven to be difficult. The current work addresses this issue by combining submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for our measurements. Exposure of the protein to a brief pulse of hydroxyl radical (·OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using mass spectrometry-based peptide mapping, providing spatially resolved measurements of changes in solvent accessibility. The submillisecond mixer used here improves the time resolution by a factor of 50 compared to earlier ·OH labeling experiments from our laboratory. Data obtained in this way indicate that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed A(E)G(H) scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. Major conformational changes go to completion within 0.1 s. The technique introduced here is capable of providing in-depth structural information on very short time scales that have thus far been dominated by low resolution (global) spectroscopic probes. By employing submillisecond mixing in conjunction with slower mixing techniques, it is possible to observe complete folding pathways, from fractions of a millisecond all the way to minutes.
- Subjects :
- Animals
Free Radicals
Horses
Mass Spectrometry methods
Oxidation-Reduction
Protein Folding
Protein Structure, Secondary
Time Factors
Apoproteins analysis
Apoproteins chemistry
Myoglobin analysis
Myoglobin chemistry
Spectrometry, Mass, Electrospray Ionization methods
Tandem Mass Spectrometry methods
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 85
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 23841479
- Full Text :
- https://doi.org/10.1021/ac401148z