Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding
| Autor: | Daphne H. E. W. Huberts, Simon Lindhoud, Adrie H. Westphal, Ruchira Engel, Sanne M. Nabuurs, Antonie J. W. G. Visser, Carlo P. M. van Mierlo |
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| Přispěvatelé: | Structural Biology |
| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Circular dichroism
Protein Folding refractive-index Flavodoxin creatine-kinase Biochemie Fluorescence correlation spectroscopy fluorescence correlation spectroscopy Biochemistry chemistry.chemical_compound Escherichia coli hydrogen-exchange HSP70 Heat-Shock Proteins Guanidine Molecular Biology Protein secondary structure inclusion-body formation Azotobacter vinelandii EPS-1 Chemistry Escherichia coli Proteins molecular chaperones Cell Biology Molten globule Recombinant Proteins Crystallography Förster resonance energy transfer protein stability escherichia-coli Protein folding Macromolecular crowding Apoproteins azotobacter-vinelandii apoflavodoxin self-association |
| Zdroj: | Journal of Biological Chemistry, 283(41), 27383-27394 Engel, R, Westphal, A H, Huberts, D H E W, Nabuurs, S M, Lindhoud, S, Visser, A J W G & Van Mierlo, C P M 2008, ' Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin unfolding. ', Journal of Biological Chemistry, vol. 283, pp. 27383-27394 . https://doi.org/10.1074/jbc.M802393200 Journal of Biological Chemistry 283 (2008) 41 Journal of Biological Chemistry, 283, 27383-27394. American Society for Biochemistry and Molecular Biology Inc. |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.M802393200 |
| Popis: | To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 +/- 3 angstrom at 3M guanidine hydrochloride. Forster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding. |
| Databáze: | OpenAIRE |
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