Moisturizing efficiency of silk protein hydrolysate: Silk fibroin

115-121A biomimetic approach of composition and natural function of natural moisturizing factor (NMF) with the amino acid content of silk fibroin was advantageously used to reconstruct the skin moisturizing system. The isolation of silk hydrolysate with water and sodium chloride treatment was complete in one hour. Lithium ion from LiBr effectively penetrated crystal domains of fibroin and gave desired solubility. Silk fibroin from Bombyx mori cocoons was non-allergic and biocompatible in skin and rabbit eye tests. The concentration dependent moisturizing efficacy of fibroin (1-5% w/v) in solution and cream form has been demonstrated by TEWL in vitro and in volunteers. As compared to dry and normal skin the fibroin containing cream revealed increased substantivity. The increased hydroxproline content was responsible for retaining higher moisture in the skin. This in turn maintained the skin in soft and supple state. The significant drop in impedance was observed within 1 hr of the application of fibroin and the effect was sustained for more than 6 hrs. Thus, increased hydration level in stratum corneum was achieved by fibroin treatment. The SEM of fibroin treated skin replicas showed a desired attribute of soft, smooth skin texture and improved flexibility. The increased state of hydration caused interdigitating of cell edges as evident in microphotographs. The rapid and sustained moisturizing efficiency observed with silk fibroin was well substantiated by the results of skin substantivity and impedance tests

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR–MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein–protein recognition needed for an efficient electron transfer process

Structure of a Baculovirus Sulfhydryl Oxidase, a Highly Divergent Member of the Erv Flavoenzyme Family ▿

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds

An electron-transfer path through an extended disulfide relay system: the case of the redox protein ALR

The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in ALR