Photoreduction of ferricytochrome c in the presence of potassium ferrocyanide.

Ferricytochrome c has been previously shown to photoreduce in the presence of ferrocyanide anions, but the process has been poorly understood. Here, we propose a kinetic model for this phenomenon and support it with resonance Raman measurements that show a clear dependence of photoreduction on the concentration of mixture components and laser power. Our data are consistent with the photoreduction process that is driven by photoexcitation of the heme followed by an electron transfer from the excited state of the heme macrocycle to the heme iron. Concomitantly, the hole in the heme ground state is filled with an electron transferred from a ferrocyanide ion residing in the heme pocket.

Ferrocyanide-Mediated Photoreduction of Ferricytochrome†C Utilized to Selectively Probe Non-native Conformations Induced by Binding to Cardiolipin-Containing Liposomes.

Ferricytochrome c binding to cardiolipin-containing liposomes produces a heterogeneous distribution of conformations comprising native-like and non-native misfolded proteins. We utilized the photoreduction of native ferricytochrome c in the presence of potassium ferrocyanide and resonance Raman spectroscopy to probe the population of native and misfolded cytochrome c on liposomes with 20 % tetraoleylcardiolipin (TOCL)/80 % dioleylphosphocholine (DOPC) and with 100 % TOCL as a function of TOCL concentration. Our data provided strong support for an earlier model, which predicts that the equilibrium between native and non-native conformations is shifted to the latter with decreasing protein occupation of liposomes.

Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.

Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-ß-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.

pH-Induced Switch between Different Modes of Cytochrome c Binding to Cardiolipin-Containing Liposomes.

Fluorescence, visible circular dichroism (CD), absorption, and resonance Raman spectroscopy techniques were combined to explore structural changes of ferricytochrome c upon its binding to cardiolipin-containing liposomes (20% 1,1',1,2'-tetraoleyolcardiolipin and 1,2-deoleyol-sn-glycero-3-phosphocholine) at acidic pH (6.5). According to the earlier work of Kawai [J. Biol. Chem.2005, 280, 34709-347171],cytochrome c binding at this pH is governed by interactions between the phosphate head groups of cardiolipin and amino acid side chains of the so-called L-site, which contains the charged residues K22, K25, K27, and potentially H26 and H33. We found that L-site binding causes a conformational transition that involves a change of the protein's ligation and spin state. In this paper, we report spectroscopic responses to an increasing number of cardiolipin-containing liposomes at pH 6.5 in the absence and presence of NaCl. The latter was found to mostly inhibit protein binding already with 50 mM concentration. The inhibition effect can be quantitatively reproduced by applying the electrostatic theory of Heimburg [Biophys. J.1995, 68, 536-546]. A comparison with corresponding spectroscopic response data obtained at pH 7.4 reveals major differences in that the latter indicates hydrophobic binding, followed by an electrostatically driven conformational change. Visible CD data suggest that structural changes in the heme pocket of liposome-bound ferricytochrome c resemble to some extent those in the denatured protein in urea at neutral and acidic pH. The measured noncoincidence between absorption and CD Soret band of cytochrome c in the presence of a large access of cardiolipin is caused by the electric field at the membrane surface. The very fact that its contribution to the internal electric field in the heme pocket is detectable by spectroscopic means suggests some penetration of the protein into membrane surface.

pH Dependence of Ferricytochrome c Conformational Transitions during Binding to Cardiolipin Membranes: Evidence for Histidine as the Distal Ligand at Neutral pH.

The conformational changes of ferricytochrome c upon binding to cardiolipin-containing small unilamellar vesicles were studied at slightly acidic pH using fluorescence, visible circular dichroism, UV-visible absorption, and resonance Raman spectroscopy. The obtained spectroscopic response data suggest a mode of interaction, which is clearly distinct from the binding process observed at neutral pH. Evidence of a reversible and electrostatic binding mechanism under these conditions is provided through binding inhibition in the presence of 150 mM NaCl. Moreover, UV-visible absorption and resonance Raman spectra reveal that the conformational ensemble of membrane bound cytochrome c is dominated by a mixture of conformers with pentacoordinated and hexacoordinated high-spin heme irons, which contrast with the dominance of low-spin species at neutral pH. While our results confirm the L-site binding proposed by Kawai et al., they point to the protonation of a histidine ligand (H33) as conformational trigger.