Bovine Serum Albumin Bends Over Backward to Interact with Aged Plastics: A Model for Understanding Protein Attachment to Plastic Debris.

Plastic pollution, a major environmental crisis, has a variety of consequences for various organisms within aquatic systems. Beyond the direct toxicity, plastic pollution has the potential to absorb biological toxins and invasive microbial species. To better understand the capability of environmental plastic debris to adsorb these species, we investigated the binding of the model protein bovine serum albumin (BSA) to polyethylene (PE) films at various stages of photodegradation. Circular dichroism and fluorescence studies revealed that BSA undergoes structural rearrangement to accommodate changes to the polymer's surface characteristics (i.e., crystallinity and oxidation state) that occur as the result of photodegradation. To understand how protein structure may inform docking of whole organisms, we studied biofilm formation of bacteriaShewanella oneidensison the photodegraded PE. Interestingly, biofilms preferentially formed on the photodegraded PE that correlated with the state of weathering that induced the most significant structural rearrangement of BSA. Taken together, our work suggests that there are optimal physical and chemical properties of photodegraded polymers that predict which plastic debris will carry biochemical or microbial hitchhikers.

Quantification of Polymer Surface Degradation Using Fluorescence Spectroscopy.

One solution to minimizing plastic pollution is to improve reuse and recycling strategies. Recycling, however, is limited by the overall degradation of plastics being used, and current techniques for monitoring this plastic degradation fail to observe this in its early stages, which is key for optimizing reusability. This research seeks to develop an inexpensive, reproducible, and nondestructive technique for monitoring degradation of polyethylene (PE) and polypropylene (PP) materials using Nile red as a fluorescent probe. Changes in Nile red's fluorescence spectra were observed upon exposure to stained, aged PE and PP samples. As the surface hydrophobicity of the plastic decreases, Nile red's fluorescence signal undergoes a corresponding signal shift to longer wavelengths (lower energy). The trends seen in the fluorescent profile were related to more commonly used measurements of plastic degradation, namely, the carbonyl index from infrared spectroscopy and bulk crystallinity from calorimetry. Results demonstrate clear trends in fluorescence spectra shifts as related to the chemical and physical changes to the plastics, with trends dependent on the polymer type but independent of polymer film thickness. The strength of this technique is divided into two defined fits of the fluorescence signal; one fit characterizes the degradation throughout the whole range of degradative oxidation and the other is tailored to provide insight into the early stages of degradation. Overall, this work establishes a characterization tool that assesses the extent of plastics' degradation, which may ultimately impact our ability to recover plastics and minimize plastic waste.

Curvature-Dependent Binding of Cytochrome c to Cardiolipin.

Cytochrome c binds cardiolipin on the concave surface of the inner mitochondrial membrane, before oxidizing the lipid and initiating the apoptotic pathway. This interaction has been studied in vitro, where mimicking the membrane curvature of the binding environment is difficult. Here we report binding to concave, cardiolipin-containing, membrane surfaces and compare findings to convex binding under the same conditions. For binding to the convex outer surface of cardiolipin-containing vesicles, a two-step structural rearrangement is observed with a small rearrangement detectable by Soret circular dichroism (CD) occurring at an exposed lipid-to-protein ratio (LPR) near 10 and partial unfolding detectable by Trp59 fluorescence occurring at an exposed LPR near 23. On the concave inner surface of cardiolipin-containing vesicles, the structural transitions monitored by Soret CD and Trp59 fluorescence are coincident and occur at an exposed LPR near 58. On the concave inner surface of mitochondrial cristae, we estimate the LPR of cardiolipin to cytochrome c is between 50 and 100. Thus, cytochrome c may have adapted to its native environment so that it can undergo a conformational change that switches on its peroxidase activity when it binds to CL-containing membranes in the cristae early in apoptosis. Our results show that membrane curvature qualitatively affects peripheral protein-lipid interactions and also highlights the disparity between in vitro binding studies and their physiological counterparts where cone-shaped lipids, like cardiolipin, are involved.

The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis.

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.

Cardiolipin Preferentially Partitions to the Inner Leaflet of Mixed Lipid Large Unilamellar Vesicles.

Cardiolipin (CL), an anionic phospholipid constituting 20% of the inner mitochondrial membrane (IMM) of eukaryotes, stabilizes electron transport chain (ETC) complexes and is a signaling agent in the early stages of apoptosis. For apoptosis, CL moves from the inner to the outer leaflet of the IMM via a poorly understood mechanism. Relative to cylindrically shaped lipids like dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG), cone-shaped CL should prefer the concave surfaces of lipid bilayers. Using the fluorophore, 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)phenyl]ethene, we have measured CL versus DOPG partitioning to the inner versus the outer leaflet of liposomes in mixed lipid systems with DOPC. DOPG shows no leaflet preference. However, CL has a 4:1 preference for the concave surface of the inner leaflet of liposomes. To further test the inner leaflet preference of CL, we show that cytochrome c binding to the outer convex surface of 20% CL/80% DOPC vesicles is strongly attenuated. Because the outer leaflet of intracristal regions of the IMM has a concave curvature, the preference of CL for concave surfaces may facilitate the transport of CL from the inner to the outer leaflet of the IMM needed for CL remodeling, the optimal functioning of the ETC, and signaling in the early stages of apoptosis.

Electrostatic Constituents of the Interaction of Cardiolipin with Site A of Cytochrome c.

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.

Rapid quantification of vesicle concentration for DOPG/DOPC and Cardiolipin/DOPC mixed lipid systems of variable composition.

A novel approach to quantify mixed lipid systems is described. Traditional approaches to lipid vesicle quantification are time consuming, require large amounts of material and are destructive. We extend our recently described method for quantification of pure lipid systems to mixed lipid systems. The method only requires a UV-Vis spectrometer and does not destroy sample. Mie scattering data from absorbance measurements are used as input into a Matlab program to calculate the total vesicle concentration and the concentrations of each lipid in the mixed lipid system. The technique is fast and accurate, which is essential for analytical lipid binding experiments.

Site A-Mediated Partial Unfolding of Cytochrome c on Cardiolipin Vesicles Is Species-Dependent and Does Not Require Lys72.

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.

Rapid quantification of cardiolipin and DOPC lipid and vesicle concentration.

A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration. Fast and accurate concentration determination for preparations of vesicles is essential for analytical titration experiments necessary for protein/vesicle binding curves.