45 research outputs found
Population Analysis of Folding Intermediates From Time-Resolved and Spectral Fluorescence of Single-Tryptophan Apoflavodoxin
Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement
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 α–β 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 α-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
Molecular Dynamics Study of the Solvation of an α-Helical Transmembrane Peptide by DMSO
Formation of On- and Off-Pathway Intermediates in the Folding Kinetics of Azotobacter vinelandii
Identifying, exploring and integrating the spiritual dimension in proactive care planning: A mixed methods evaluation of a communication training intervention for multidisciplinary palliative care teams.
Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein
Upon adding denaturant, apoflavodoxin’s donor emission spectrum and acceptor excitation spectrum shift to the red.
<p>(a) Normalized emission spectra of ‘donor-only’ apoflavodoxin. The inset zooms in on the fluorescence emission maximum, which shifts from 518 to 521 nm upon adding 6.9 M GuHCl. (b) Normalized excitation spectrum of acceptor of d69-a178. GuHCl concentrations are 0.1 M (solid line), 1.7 M (dotted line) and 6.9 M (dashed line), respectively.</p
Distances between residues used in dye-labeling and tryptophans of native apoflavodoxin.
<p>The distances reported are between C<sup>7a</sup> of the tryptophan indicated and C<sup>α</sup> of the residue to be dye-labeled, as measured by using PyMol (Schrödinger, LLC, Palo Alto, Ca, USA) and the crystal structure of <i>A. vinelandii</i> flavodoxin (pdb ID 1YOB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045746#pone.0045746-Alagaratnam1" target="_blank">[56]</a>).</p
Denaturant-dependencies of the reorientation rates of dye labels attached to apoflavodoxins.
<p>Shown are the <i>D</i><sub>⊥</sub> data of A488 of ‘donor-only’ protein (open circles), and of A568 of d69-a1 (blue circles), d69-a131 (green diamonds) and d69-a178 (orange squares), respectively.</p