Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control

Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar ‘superfolded’ proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues

Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins

The Corrosion Studies of Electron Beam Welded Nickel Alloy

The paper presents the influence of electron - beam (EB) remelting effect on the surface layer electrochemical parameters obtained from potentiodynamic anodic polarization studies and impedance spectroscopy measurements for a set of Inconel 617 electron beam remelted obtained for different process parameters. The correlation between EBW process parameters and characteristic of surface oxide layer properties and resistance to the acidic environment were discussed. The electrochemical studies were supported by microstructural analysis of the remelted zone (RZ), heat affected zone (HAZ), native metal and observed precipitates formed under rapid solidification process. Both electrochemical technics applied to evaluate corrosion properties of remelted Inconel 617 evidenced a strong influence of the electron beam current on the corrosion resistance

The Corrosion Studies of Electron Beam Welded Nickel Alloy

The paper presents the influence of electron - beam (EB) remelting effect on the surface layer electrochemical parameters obtained from potentiodynamic anodic polarization studies and impedance spectroscopy measurements for a set of Inconel 617 electron beam remelted obtained for different process parameters. The correlation between EBW process parameters and characteristic of surface oxide layer properties and resistance to the acidic environment were discussed. The electrochemical studies were supported by microstructural analysis of the remelted zone (RZ), heat affected zone (HAZ), native metal and observed precipitates formed under rapid solidification process. Both electrochemical technics applied to evaluate corrosion properties of remelted Inconel 617 evidenced a strong influence of the electron beam current on the corrosion resistance

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm