title: Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding.
abstract: 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 A at 3 M guanidine hydrochloride. Förster 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.
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Ruchira
authors.initials: R
authors.lastname: Engel
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Daphne H E W
authors.initials: DHEW
authors.lastname: Huberts
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Sanne M
authors.initials: SM
authors.lastname: Nabuurs
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: Lindhoud
authors.affiliation: Department of Structural Biology, Institute of Molecular Cell Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Antonie J W G
authors.initials: AJWG
authors.lastname: Visser
authors.affiliation: Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands. Electronic address: carlo.vanmierlo@wur.nl.
authors.forename: Carlo P M
authors.initials: CPM
authors.lastname: van Mierlo
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001059:Apoproteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; C026932:apoflavodoxin; C063842:dnaK protein, E coli