Graphical analysis of interactions between oxidation-reduction sites in two site oxidation-reduction protein

Many enzymes that catalyze electron-transfer reaction contain multiple oxidation-reduction centers (sites). The oxidation-reduction potential of one site as well as the kinetics of electron transfer through this site may be altered by the state of reduction of a neighboring site. Oxidation-reduction site interactions may be mechanistically important and quantitation of site interactions would aid the interpretation of thermodynamic data and possibly kinetic data. A graphical means to detect and quantitate interactions between oxidation-reduction sites from oxidation-reduction equilibrium data (type A + B C + D)is described and has its roots in the Scatchard analysis of ligand binding equilibria (type A + B C). Oxidation-reduction sites often have distinct physical properties allowing the titration behavior of specific sites to be monitored. Equilibrium measurements on specific sites of a two site protein allow a further analysis of the data which can be combined with the oxidation-reduction Scatchard analysis to solve for all four specific site equilibrium constants. Ligand binding systems can usually measure only total site binding and simplifying assumptions of identical sites or noninteracting sites are required to solve for the site specific equilibrium constants. Thus, specific site equilibrium measurements offer a distinct advantage over total site measurements. The principles of the method are illustrated by applying the graphical analysis to the two site protein, thioredoxin reductase, which contains an oxidation-reduction active site disulfide in addition to FAD. The specific site oxidation-reduction midpoint potentials (Em) of the FAD and disulfide couples of thioredoxin reductase at pH 6.0, 12[deg]C, were found to be FAD/FADH2-enzyme-(S)2 = -0.183 V, FAD/FADH2-enzyme-(SH)2 = -0.199 V, (FAD)-enzyme-(S)2/(SH)2 = -0.202 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.218 V. Hence, at pH 6.0, the FAD and disulfide sites of thioredoxin reductase have Em values that differ by approximately 0.019 V and have a negative interaction of about 0.016 V.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24992/1/0000419.pd

Protein disulphide isomerase-mediated grafting of cysteine-containing peptides onto over-bleached hair

The ability of Protein disulphide isomerase (PDI) to promote the grafting of two cysteine-containing peptides onto hair was investigated in order to develop an alternative treatment for over-bleached hair. The studied peptides were designed based on human keratin and human lung surfactant proteins and were linked to a fluorescent dye to facilitate visualisation of the grafting process and to assess hair penetration. The ability of the peptides to restore mechanical and thermal properties lost by repeated bleaching treatments was also studied. After eight bleaching treatments, hair samples displayed 42% less mechanical resistance, coupled with a decrease in α-helix denaturation enthalpies and temperatures. Hair surface damage following bleaching was visualized by scanning electron microscopy. Addition of PDI to the treatment formulations promoted peptide attachment to the hair via disulphide bonds, facilitating their penetration into the hair cortex, as observed by fluorescence microscopy. The proposed peptide treatment resulted in an increase in α-helix denaturation enthalpy in over-bleached hair, as well as an increase in both Young's modulus and tensile strength. Thus, mechanical and thermal properties were improved after the peptide treatment in the presence of PDI; suggesting that the formulations presented in this work are promising candidates for hair-care applications

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.We thank to FCT 'Fundacao para a Ciencia e Tecnologia' (scholarship SFRH/BD/38363/2007) for providing Margarida Fernandes the grant for PhD studies