Measurements at pH 8 allow evaluation of binding of 100% cardiolipin vesicles to site A of cytochrome c without interference from other known binding sites. Site A encompasses Lys72, Lys73, Lys86, and Lys87, located in or adjacent to Ω-loop D (residues 70–85), which positions Met80 for binding to the heme. Binding of cytochrome c to cardiolipin disrupts Met80 heme binding, permitting peroxidase activity. Binding of cardiolipin to yeast iso-1-cytochrome c versus human cytochrome c is compared to assess how binding of cardiolipin to site A has evolved for cytochrome c from species that do not have a complete intrinsic apoptotic pathway to species that do. Using a nondestructive method of quantifying cardiolipin concentration, highly reproducible binding curves are obtained. The results indicate two sequential structural rearrangements on the surface of 100% cardiolipin vesicles. The first, more modest, structural rearrangement occurs at an exposed (outer leaflet) lipid:protein ratio of 8–10 for both cytochromes c. The second, occurring at higher lipid:protein ratios, causes significant unfolding of cytochrome c and requires a much higher lipid:protein ratio for human versus yeast cytochrome c. Higher lipid:protein ratios enhance the peroxidase activity of cytochrome c, suggesting that human cytochrome c has evolved a more stringent on/off switch for cardiolipin peroxidation in the early stages of apoptosis. For both human and yeast cytochrome c, the K72A mutation has only minor effects on binding to site A, suggesting that other nearby lysines can compensate for the lack of Lys72.

Cytochrome c binds to cardiolipin (CL) on the inner mitochondrial membrane during the initial stages of apoptosis where it oxidizes CL, promoting its release into the cytoplasm where it initiates apoptosis. Previous work has identified interaction sites on cytochrome c involved in the cytochrome c-CL interaction. The contributions of the lysines attributed to site A, the anionic site, are studied here to elucidate the relative importance of each for electrostatic interaction of cytochrome c with CL at pH 8, conditions where site A is dominant. A set of single, double, and quadruple lysine to alanine variants of yeast iso-1-cytochrome c, at sequence positions 72, 73, 86, and 87, show that all contribute to the site A-mediated interaction with CL. All variants experience two sequential structural rearrangements as the lipid to protein ratio (LPR) increases. At a low LPR near 10, all variants undergo a small heme-centered structural change detected by Soret circular dichroism. At higher LPRs ranging from 22 to 34, all variants partially unfold as detected by Trp59 emission. The robustness of the mechanism of interaction to sequential neutralization of the four lysines assigned to site A demonstrates that site A is more extensive than previously supposed. The nature of both structural rearrangements also depends on which lysines constitute site A. The peroxidase activity of cytochrome c in the early stages of apoptosis depends on the nature of structural rearrangement near the heme. Thus, the lysines that comprise site A may have evolved to optimize the peroxidase signaling switch.

Understanding the degradation of plastics, some of the most widely used materials on Earth, is crucial in a broad range of fields from materials design to environmental monitoring. Many polymers yellow as they age, but there is no chemical explanation that can describe the origin of this yellowing for polyolefins specifically. Here, we show that irradiated blown polyethylene sheets preferentially scatter circularly polarized light. Because scattering of circularly polarized light only occurs in the presence of chiral structures, our findings provide evidence of formation of chiral supramolecular structures responsible for preferential light scattering that may be the underpinnings for the perceived yellow/ brown tint as polyethylene ages. Further, we demonstrate incident polarization-dependent detection of colored light scattering from irradiated polyethylene films and that the scattered light is distinctly different in color. Overall, these results provide evidence that the yellowing of polyethylene, previously assumed to be caused by polymer backbone rearrangements, is actually the product of chiral, optically active structures that form on the plastic's surface due to UV irradiation. To the best of our knowledge, this is the first explanation for polymer discoloring that provides evidence for the development of supramolecular structures of polymers during aging. Because of this, our findings provide an alternative direction in plastic degradation research for understanding the chemical and structural changes. Findings presented here shift our understanding about materials degradation and can inform our future materials designs and recovery.

Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of β-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.

Oxidation of cardiolipin (CL) by cytochrome c (cytc) has been proposed to initiate the intrinsic pathway of apoptosis. Domain-swapped dimer (DSD) conformations of cytc have been reported both by our laboratory and by others. The DSD is an alternate conformer of cytc that could oxygenate CL early in apoptosis. We demonstrate here that the cytc DSD has a set of properties that would provide tighter regulation of the intrinsic pathway. We show that the human DSD is kinetically more stable than horse and yeast DSDs. Circular dichroism data indicate that the DSD has a less asymmetric heme environment, similar to that seen when the monomeric protein binds to CL vesicles at high lipid:protein ratios. The dimer undergoes the alkaline conformational transition near pH 7.0, 2.5 pH units lower than that of the monomer. Data from fluorescence correlation spectroscopy and fluorescence anisotropy suggest that the alkaline transition of the DSD may act as a switch from a high affinity for CL nanodiscs at pH 7.4 to a much lower affinity at pH 8.0. Additionally, the peroxidase activity of the human DSD increases 7-fold compared to that of the monomer at pH 7 and 8, but by 14-fold at pH 6 when mixed Met80/H2O ligation replaces the lysine ligation of the alkaline state. We also present data that indicate that cytc binding shows a cooperative effect as the concentration of cytc is increased. The DSD appears to have evolved into a pH-inducible switch that provides a means to control activation of apoptosis near pH 7.0.