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 Å 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°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.

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

“…The Molten Globule Folding Intermediate of Flavodoxin Is Almost Entirely Structured-The hydrodynamic radius of the molten globule of flavodoxin is expanded only by about 11% compared with the one of native apoflavodoxin, as demonstrated by fluorescence correlation spectroscopy experiments (30). The molten globule is thus relatively compact.…”

“…Spectroscopic data show that the off-pathway folding species of flavodoxin is molten globule-like; its hydrodynamic radius is closer to the native state than to the unfolded state, its three tryptophans are solvent-exposed, and it has severely broadened NMR resonances due to exchange between different conformers on the micro-to millisecond time scale (18,29,30). An off-pathway intermediate is experimentally observed for all other ␣-␤ parallel proteins of which the kinetic folding has been investigated (i.e.…”

“…We showed that at biologically relevant protein concentrations or by mimicking molecular crowding conditions present in cells, severe aggregation of the flavodoxin off-pathway species occurs (29,30). To further the understanding of the conformational properties of the flavodoxin molten globule, in this study, H/D exchange detected by NMR spectroscopy is used as a powerful technique that gives detailed information about a protein at the level of single amino acids.…”

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

“…Achieving and maintaining the correct three‐dimensional protein structure is a continuous struggle within cells. Firstly, folding of proteins toward an active biological state is challenged by the crowded environment within the cell, which may lead to off‐pathway reactions resulting in protein aggregation (Ellis & Minton, 2006; Engel et al ., 2008; Homouz et al ., 2008). Protein misfolding can further originate from direct protein damage (e.g., oxidation, thermal denaturation), but can also originate from age‐related mutations, molecular misreading (van Leeuwen et al ., 2000), splicing errors (Pettigrew & Brown, 2008), or errors in translation (Parker, 1989; Kramer & Farabaugh, 2007).…”

Low crowding agent concentration destabilizes against pressure unfolding

“…On the contrary the addition of high concentrations of different polymers dramatically accelerated alpha-synuclein fibrillation in vitro depending on the nature, length and concentration of the polymer [8]. Crowding enhances aggregation propensity of several proteins like apoflavodoxin, creatine kinase [9,10], but almost completely inhibits amyloid formation of lysozyme and stabilizes lysozyme activity [1,11]. The paradoxical results can be understood probably by recalling, that aggregation needs some kind of destabilization of the protein.…”