Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode.

Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0-90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV-Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

Associations of HIV Testing, Sexual Risk and Access to Prevention Among Female Sex Workers in the Dominican Republic.

The Caribbean region has one of the highest proportions of HIV in the general female population attributable to sex work. In 2008 (n = 1256) and 2012 (n = 1525) in the Dominican Republic, HIV biological and behavioral surveys were conducted among female sex workers (FSW) in four provinces using respondent driven sampling. Participants were ≥15 years who engaged in intercourse in exchange for money in the past 6 months and living/working in the study province. There were no statistically significant changes in HIV and other infections prevalence from 2008 to 2012, despite ongoing risky sexual practices. HIV testing and receiving results was low in all provinces. FSW in 2012 were more likely to receive HIV testing and results if they participated in HIV related information and education and had regular checkups at health centers. Further investigation is needed to understand barriers to HIV testing and access to prevention services.

Perturbation of the redox site structure of cytochrome c variants upon tyrosine nitration.

Post-translational nitration of tyrosine is considered to be an important step in controlling the multiple functions of cytochrome c (Cyt-c). However, the underlying structural basis and mechanism are not yet understood. In this work, human Cyt-c variants in which all but one tyrosine has been substituted by phenylalanine have been studied by resonance Raman and electrochemical methods to probe the consequences of tyrosine nitration on the heme pocket structure and the redox potential. The mutagenic modifications of the protein cause only subtle conformational changes of the protein and small negative shifts of the redox potentials which can be rationalized in terms of long-range electrostatic effects on the heme. The data indicate that the contributions of the individual tyrosines for maintaining the relatively high redox potential of Cyt-c are additive. Nitration of individual tyrosines leads to a destabilization of the axial Fe-Met80 bond which causes the substitution of the native Met ligand by a water molecule or a lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of the heme pocket of Cyt-c due to tyrosine nitration and to local electric fields are independent of each other and occur via different molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the peroxidase function.

Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119.

Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.