Efficient Oxidation and Destabilization of Zn(Cys)4 Zinc Fingers by Singlet Oxygen

Singlet oxygen (¹O₂) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)₄ zinc finger with ¹O₂ by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by ¹O₂. The reaction of the ZnS₄ core with ¹O₂ is very fast and efficient with almost no physical quenching of ¹O₂. A drastic (ca. five orders of magnitude) decrease of the Zn²⁺ binding constant was observed upon oxidation. This suggests that the Zn(Cys)₄ zinc finger proteins would release their Zn²⁺ ion and unfold upon reaction with ¹O₂ under cellular conditions and that zinc finger sites are likely targets for ¹O₂.     

Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de?novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in?vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster.     

Determination of peptide-surface adsorption free energy for material surfaces not conducive to SPR or QCM using AFM

The interactions between peptides and proteins with material surfaces are of primary importance in many areas of biotechnology. While surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance (QCM) methods have proven to be very useful in measuring fundamental properties characterizing adsorption behavior, such as the free energy of adsorption for peptide-surface interactions, these methods are largely restricted to use for materials that can readily form nanoscale-thick films over the respective sensor surfaces. Many materials including most polymers, ceramics, and inorganic glasses, however, are not readily suitable for use with SPR or QCM methods. To overcome these limitations, we recently showed that desorption forces (F(des)) obtained using a standardized AFM method linearly correlate to standard-state adsorption free energy values (ΔG°(ads)) measured from SPR in phosphate buffered saline (PBS: phosphate buffered 140 mM NaCl, pH 7.4). This approach thus provides a means to determine ΔG°(ads) for peptide adsorption using AFM that can be applied to any flat material surface. In this present study, we investigated the F(des)-ΔG°(ads) correlation between AFM and SPR data in PBS for a much broader range of systems including eight different types of peptides on a set of eight different alkanethiol self-assembled monolayer (SAM) surfaces. The resulting correlation was then used to estimate ΔG°(ads) from F(des) determined by AFM for selected bulk polymer and glass/ceramic materials such as poly(methyl methacrylate) (PMMA), high-density polyethylene (HDPE), fused silica glass, and a quartz (100) surface. The results of these studies support our previous findings regarding the strong correlation between F(des) measured by AFM and ΔG°(ads) determined by SPR, and provides a means to estimate ΔG°(ads) for peptide adsorption on macroscopically thick samples of materials that are not conducive for use with SPR or QCM.     

Time-dependent conformational changes in adsorbed albumin and its effect on platelet adhesion

Recent studies have shown that platelets can adhere to adsorbed albumin (Alb) through a receptor-mediated mechanism, but only if the Alb undergoes more than a critical degree of adsorption-induced unfolding. The objectives of this research were to investigate whether Alb that was initially adsorbed in a manner that induced unfolding that was less than this critical level would undergo further unfolding with time and, if so, whether this would induce the onset of platelet adhesion once this critical level was exceeded. To address these questions, CD spectropolarimetry was used to monitor the structure of Alb on OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces, with the Alb initially adsorbed under conditions resulting in degrees of unfolding that were below this critical level, and then the adsorbed Alb layers were aged over 6 months in sterile physiological saline at 37 °C. Platelet adhesion to Alb was quantified at selected time points via a lactate dehydrogenase (LDH) assay. The results indicate that an adsorbed Alb layer does undergo further structural changes with increasing residence time and supports platelet adhesion once it unfolds beyond the previously determined critical level. These results may be relevant to the clinically observed problem of the onset of late-thrombosis, which occurs on cardiovascular implants such as drug-eluting stents.     

Selective protein adsorption on a phase-separated solvent-cast polymer blend

Polymer-based biomedical devices are growing increasingly sophisticated as compositions evolve toward copolymers and blends in order to satisfy complex design criteria. Such polymers afford opportunities for both micro- and macrophase separation at nano- and micro-length scales and raise questions concerning the role of heterogeneous surface morphology on protein adsorption. Adsorbed protein layers play a critical role in mediating the interaction of cells with polymer surfaces, and both understanding and controlling protein adsorption is assuming greater significance in the development of surfaces with enhanced physiological compatibility. Here we study the short-time adsorption of ferritin, a model protein highly resistant to denaturation and easily imaged in the transmission electron microscope (TEM), onto a phase-separated homopolymer blend of polycaprolactone (PCL) and a polycarbonate derived from desaminotyrosyl-tyrosine dodecyl ester (PDTD). At physiological pH, ferritin selectively adsorbs onto the PDTD phase at a surface density approximately three times greater than that on the PCL phase. By decreasing the pH below ferritin's isoelectric point so its average charge becomes positive, the selective adsorption disappears and the surface density of adsorbed ferritin becomes independent of the phase separation. We attribute the selectivity to the electrostatic repulsion between ferritin and hydrolytically charged PCL, both of which will have a net negative charge at physiological pH. To perform these experiments, we solvent-cast ultrathin polymer films onto dissolvable salt substrates, and we characterize the morphology by TEM imaging and quantitative spatially resolved electron energy-loss spectroscopy (EELS). We find that the film morphology depends strongly on such processing-related variables as the solvent evaporation rate and the nature of the surface in contact with the polymer film during casting. The adsorption of ferritin depends on whether the film is phase-separated as well as to which surface of the film the protein solution is exposed, and these findings suggest that seemingly small variations in polymer processing that influence both the bulk and surface morphology can have a profound effect on the short-time protein adsorption.     

X-ray photoreduction of a di(mu-oxo)Mn(III)Mn(IV) complex occurs at temperatures as low as 20 K

Full reduction of the Mn(III)(mu-O)2Mn(IV) core to Mn(II)(mu-OH2)2Mn(II) is observed upon irradiation by an X-ray beam at ca. 20 K.     

Submillimeter infrared spectroscopy of fluorinated polymers - phase transition study

Submillimeter infrared technique is used to study the phase transitions in poly(vinylidene fluoride) homopolymer and copolymers with trifluorethylene. During the phase transition form II to form I, induced by copolymerization, electrical or mechanical stresses, the spectra show an important increasing of the submillimeter absorption. This behavior is explained by changes of the polarizability at phase transition.