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.
Directing the Oligomer Size Distribution of Peroxidase-Mediated Cross-Linked Bovine a-Lactalbumin
Heijnis, W.H. ; Wierenga, P.A. ; Berkel, W.J.H. van; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)9. - ISSN 0021-8561 - p. 5692 - 5697.
tyrosine-containing peptides - microbial transglutaminase - horseradish-peroxidase - hydrogen-peroxide - molten globule - whey proteins - apo-bovine - linking - oxidation - enzyme
Enzymatic protein cross-linking is a powerful tool to change protein functionality. For optimal functionality in gel formation, the size of the cross-linked proteins needs to be controlled, prior to heating. In the current study, we addressed the optimization of the horseradish peroxidase-mediated cross-linking of calcium-depleted bovine a-lactalbumin. To characterize the formed products, the molecular weight distribution of the cross-linked protein was determined by size exclusion chromatography. At low ionic strength, more dimers of a-lactalbumin are formed than at high ionic strength, while the same conversion of monomers is observed. Similarly, at pH 5.9 more higher oligomers are formed than at pH 6.8. This is proposed to be caused by local changes in apo a-lactalbumin conformation as indicated by circular dichroism spectroscopy. A gradual supply of hydrogen peroxide improves the yield of cross-linked products and increases the proportion of higher oligomers. In conclusion, this study shows that the size distribution of peroxidase-mediated cross-linked a-lactalbumin can be directed toward the protein oligomers desired.
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
Conformational studies on a hyperthermostable enzyme
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
FEBS Journal 272 (2005)21. - ISSN 1742-464X - p. 5484 - 5496.
time-resolved fluorescence - pyrococcus-furiosus - secondary structure - circular-dichroism - tryptophan fluorescence - denatured proteins - unfolded state - molten globule - sh3 domain - c-13 nmr
The structural features of the hyperthermophilic endo-ß-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 °C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 °C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.