10 research outputs found
Extrinsic Fluorescent Dyes as Tools for Protein Characterization
Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization
The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal: DIRECT ANALYSIS BY FLUORESCENCE-DETECTED ANALYTICAL ULTRACENTRIFUGATION*
Transthyretin (TTR) is normally a stable plasma protein. However, in cases
of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR
is deposited as amyloid fibrils, leading to organ dysfunction and possibly
death. The mechanism by which TTR undergoes the transition from stable,
soluble precursor to insoluble amyloid fibril and the factors that promote
this process are largely undetermined. Most models involve the dissociation of
the native TTR tetramer as the initial step. It is largely accepted that the
TTR gene mutations associated with TTR-related amyloidosis lead to the
expression of variant proteins that are intrinsically unstable and prone to
aggregation. It has been suggested that amyloidogenicity may be conferred to
wild-type TTR (the form deposited in SSA) by chemical modification of the lone
cysteine residue (Cys10) through mixed disulfide bonds.
S-Sulfonation and S-cysteinylation are prevalent TTR
modifications physiologically, and studies have suggested their ability to
modulate the structure of TTR under denaturing conditions. In the present
study, we have used fluorescence-detected sedimentation velocity to determine
the effect of S-sulfonate and S-cysteine on the quaternary
structural stability of fluorophore-conjugated recombinant TTR under
nondenaturing conditions. We determined that S-sulfonation stabilized
TTR tetramer stability by a factor of 7, whereas S-cysteinylation
enhanced dissociation by 2-fold with respect to the unmodified form. In
addition, we report the direct observation of tetramer stabilization by the
potential therapeutic compound diflunisal. Finally, as proof of concept, we
report the sedimentation of TTR in serum and the qualitative assessment of the
resulting data