Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein
| Autor: | Jan Willem Borst, Carlo P. M. van Mierlo, Adrie H. Westphal, Simon Lindhoud |
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| Rok vydání: | 2012 |
| Předmět: |
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Molecular Protein Denaturation Protein Folding refractive-index Flavodoxin 7. Clean energy 01 natural sciences Biochemistry Protein Refolding Protein Structure Secondary state chemistry.chemical_compound Native state hydrogen-exchange Fluorescence Resonance Energy Transfer Macromolecular Structure Analysis Guanidine Protein secondary structure 0303 health sciences Multidisciplinary biology Protein Stability Physics aggregation Energy landscape Molten globule Medicine Research Article Protein Structure Science Biophysics mechanism Biochemie flavodoxin 010402 general chemistry Protein Chemistry 03 medical and health sciences fluorescence depolarization Protein Interactions Biology 030304 developmental biology Alexa Fluor VLAG Azotobacter vinelandii spectroscopic ruler Staining and Labeling energy landscape Proteins Computational Biology 0104 chemical sciences Protein Structure Tertiary Förster resonance energy transfer chemistry biology.protein azotobacter-vinelandii apoflavodoxin Apoproteins |
| Zdroj: | PLoS ONE PLoS ONE, Vol 7, Iss 9, p e45746 (2012) PLoS ONE 7 (2012) 9 PLoS ONE, 7(9) |
| ISSN: | 1932-6203 |
| Popis: | Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Forster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein. |
| Databáze: | OpenAIRE |
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