Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a ß-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of ß-2 microglobulin (ß2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between ß-sheets orthogonal to the ß-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric ß2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the ß-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.

Delineating the conformational elements responsible for Cu(2+)-induced oligomerization of beta-2 microglobulin.

Beta-2 microglobulin (beta2m) is a small globular protein implicated in amyloid fiber formation in renal patients on long-term hemodialysis therapy. In vitro, under physiological conditions, beta2m is not aggregation prone. However, in the presence of stoichiometric Cu(2+), beta2m readily self-associates ultimately leading to heterogeneously sized aggregates. As this process occurs under near physiological solution conditions where the fold is >or=20 kJ/mol stabilized over the unfolded state, local conformational rearrangements are critical to understanding the oligomerization of beta2m. The isomerization of a conserved cis proline at residue 32 is a recognized step in this process that can be initiated by Cu(2+) binding. To better understand the structural basis of metal-induced oligomerization of beta2m, we set out to determine the role of individual imidazole side chains in mediating metal binding affinity, native state stability, and oligomerization in the framework of P32A beta2m. We find that P32A in the presence of Cu(2+) forms a tetramer in an apparently cooperative manner. One interface of this tetramer appears to reside along an edge strand as H51 is a key residue in mediating oligomerization. Furthermore, H31 is the main Cu(2+) binding residue in P32A and has an important role in stabilizing the protein in its holo form. Importantly, Cu(2+) binding affinity in P32A is much greater than in WT. Here, we show that this strong binding affinity need not be directly coupled to oligomerization. We interpret our results in terms of the known structures of beta2m(apo) and a reversible hexameric state of beta2m(holo).

Endotoxin-induced mitochondrial damage correlates with impaired respiratory activity.

OBJECTIVE: This study was designed to determine whether mitochondrial function in a systemic organ is acutely impaired in a resuscitated model of sepsis (endotoxemia, lipopolysaccharide) and the relationship, if any, between this impairment and the extent of mitochondrial ultrastructural damage that occurs. DESIGN: Perspective, controlled laboratory investigation. SETTING: Animal laboratory in a university research institute. SUBJECTS: Adult male cats. INTERVENTIONS: A well-established feline model of acute endotoxemia was used wherein measures were taken to minimize tissue hypoxia. After lipopolysaccharide (3 mg/kg intravenously, n = 9) or isotonic saline vehicle (control, n = 5) administration, liver samples were obtained at 4 hrs posttreatment, and mitochondrial ultrastructure and respiratory function were assessed. Mitochondrial ultrastructural injury was graded on a scale of 0 (no injury) to 5 (severe injury), and mitochondrial respiration was evaluated by using standard techniques. MEASUREMENTS AND MAIN RESULTS: Significant mitochondrial injury was apparent by 4 hrs, but only in the lipopolysaccharide-treated group (2.5 +/- 0.2 vs. 1.3 +/- 0.2, p <.001) and despite maintenance of tissue oxygen availability. In addition, lipopolysaccharide treatment reduced the rate of state 3 (adenosine 5'-diphosphate-dependent) respiration, especially at complex IV (40% inhibition), and increased the rate of state 4 (adenosine 5'-diphosphate-independent) respiration, reflecting partial uncoupling of mitochondrial oxidative phosphorylation. Finally, a significant correlation was demonstrated between the severity of ultrastructural injury and the magnitude of mitochondrial respiratory dysfunction after lipopolysaccharide treatment and despite resuscitation efforts. CONCLUSION: Mitochondrial function is significantly impaired during acute sepsis, and this impairment is strongly associated with the extent of mitochondrial ultrastructural abnormalities present in the tissues. These findings in conjunction with those previously shown suggest that mitochondrial functional impairment may contribute to the pathogenesis of altered oxygen metabolism in systemic organs during sepsis.