Illuminating the off-pathway nature of the molten globule folding intermediate of an a-ß parallel protein

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 Förster 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 a-ß parallel protei

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxin's complex folding pathway

Fluorescence of Alexa Fluor dye tracks protein folding

Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding

Mustafa Kemal'in Boğazlara dair görüşü

Taha Toros Arşivi, Dosya Adı: Milli Mücadele İstiklal Harbi GazetesiUnutma İstanbul projesi İstanbul Kalkınma Ajansı'nın 2016 yılı "Yenilikçi ve Yaratıcı İstanbul Mali Destek Programı" kapsamında desteklenmiştir. Proje No: TR10/16/YNY/010

The Arabidopsis thaliana SERK1 kinase domain spontaneously refolds to an active state in vitro

Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state

Cofactor binding protects flavodoxin against oxidative stress

In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modificatio

Protein topology affects the appearance of intermediates during the folding of proteins with a flavodoxin-like fold.

The topology of a native protein influences the rate with which it is formed, but does topology affect the appearance of folding intermediates and their specific role in kinetic folding as well? This question is addressed by comparing the folding data recently obtained on apoflavodoxin from Azotobacter vinelandii with those available on all three other ¿-ß parallel proteins the kinetic folding mechanism of which has been studied, i.e. Anabaena apoflavodoxin, Fusarium solani pisi cutinase and CheY. Two kinetic folding intermediates, one on-pathway and the other off-pathway, seem to be present during the folding of proteins with an ¿-ß parallel, also called flavodoxin-like, topology. The on-pathway intermediate lies on a direct route from the unfolded to the native state of the protein involved. The off-pathway intermediate needs to unfold to allow the production of native protein. Available simulation data of the folding of CheY show the involvement of two intermediates with characteristics that resemble those of the two intermediates experimentally observed. Apparently, protein topology governs the appearance and kinetic roles of protein folding intermediates during the folding of proteins that have a flavodoxin-like fold