Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Lindhoud, S. ; Westphal, A.H. ; Mierlo, C.P.M. van; Visser, A.J.W.G. ; Borst, J.W. - \ 2014
International Journal of Molecular Sciences 15 (2014)12. - ISSN 1661-6596 - p. 23836 - 23850.
resonance energy-transfer - beta parallel protein - single-molecule fluorescence - azotobacter-vinelandii - spectroscopic ruler - refractive-index - tryptophan residue - molten globule - wild-type - pathway
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
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203 - 8 p.
azotobacter-vinelandii apoflavodoxin - resonance energy-transfer - beta parallel protein - molten-globule state - flavodoxin-ii - molecules - pathway - chains - intermediate - spectroscopy
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.
Cofactor binding protects flavodoxin against oxidative stress
Lindhoud, S. ; Berg, W.A.M. van den; Heuvel, R.H.H. van den; Heck, A.J.R. ; Mierlo, C.P.M. van; Berkel, W.J.H. van - \ 2012
PLoS ONE 7 (2012)7. - ISSN 1932-6203
methionine sulfoxide reductase - azotobacter-vinelandii apoflavodoxin - para-hydroxybenzoate hydroxylase - alkyl hydroperoxide reductase - cysteine sulfinic acid - beta parallel protein - sulfenic acid - pseudomonas-fluorescens - hydrogen-peroxide - mass-spectrome
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 modification
Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement
Nabuurs, S.M. ; Kort, B.J. de; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 689 - 698.
beta parallel protein - azotobacter-vinelandii apoflavodoxin - denatured state - folding mechanism - hydrogen-exchange - molten globule - flavodoxin-ii - native-like - pathway - nmr
Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this a–ß parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native a-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin