Protein trans-splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non-native exteins in Escherichia coli. Npu DnaE possesses robust trans-splicing activity with an efficiency of >98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N-and C-terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans-splicing activity. Protein splicing with the Npu DnaE N is also more tolerant of amino acid substitutions in the C-terminal extein sequence.

The photothermal conversion of CO2 provides a straightforward and effective method for the highly efficient production of solar fuels with high solar-light utilization efficiency. This is due to several crucial features of the Group VIII nanocatalysts, including effective energy utilization over the whole range of the solar spectrum, excellent photothermal performance, and unique activation abilities. Photothermal CO2 reaction rates (mol h(-1) g(-1)) that are several orders of magnitude larger than those obtained with photocatalytic methods (μmol h(-1) g(-1)) were thus achieved. It is proposed that the overall water-based CO2 conversion process can be achieved by combining light-driven H2 production from water and photothermal CO2 conversion with H2. More generally, this work suggests that traditional catalysts that are characterized by intense photoabsorption will find new applications in photo-induced green-chemistry processes.

With over 60,000 protein structures available in the Protein Data Bank, it is frequently possible use one of them to obtain starting phase information and to solve new crystal structures. Molecular replacement1–4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5–8, have allowed the rapid solution of large numbers of protein structures. Despite extensive work9–14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modeling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modeling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction datasets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction datasets of better than 3.2Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

We have cyclized the polypeptide backbone of L Llactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent relinearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of L Llactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidaseresistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N-and C-termini in close proximity.z 1999 Federation of European Biochemical Societies.

Recently, graphitic carbon nitride (g-C 3 N 4 ) has been investigated as a photocatalyst for water splitting and organic dye degradation. In this study, we have developed a simple soft-chemical method of doping Zn into g-C 3 N 4 to prepare a metal-containing carbon nitride. The doping was confirmed by x-ray photoelectron spectroscopy, and diffusion reflectance spectra revealed a significant red shift in the absorption edge of Zn/g-C 3 N 4 . This hybrid material shows high photocatalytic activity and good stability for hydrogen evolution from an aqueous methanol solution under visible light irradiation (λ 420 nm). The hydrogen evolution rate was more than 10 times higher for a 10%-Zn/g-C 3 N 4 sample (59.5 µmol h −1 ) than for pure g-C 3 N 4 . The maximum quantum yield was 3.2% at 420 nm.

Transition metal doping has always suffered from the disadvantage of reduction of photocatalytic activity because the dopant ions can form a discrete level in the forbidden band of the photocatalyst resulting in low mobility of electrons and holes in the dopant level and thus lowered activity. However, in this study the photocatalytic activity of an efficient visible-light sensitive photocatalyst, BiVO4, for water oxidation or organic compound degradation was found to be remarkably enhanced by molybdenum doping. The role of molybdenum doping in enhancing the photocatalytic activity of BiVO4 was investigated and discussed based on the changes of the surface acidity of the photocatalyst.

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.

A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain. Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales. There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins. Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 M guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cisproline) had disappeared. Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.All natural proteins known so far are linear chains of amino acids that fold into a unique three-dimensional structure dictated by the sequence of amino acids. A cyclic backbone structure has been found and synthetically introduced in small peptides, but is an almost unexplored topic in protein chemistry and protein structural research, in particular for larger proteins. The circular topology is also expected to lead to improved stability due to the reduced conformational entropy in the denatured state, according to polymer theory (1). It has indeed been shown experimentally that cyclized ␤-lactamase was stabilized against heat precipitation and exopeptidase degradation (2) and that the cyclization also improved in vitro thermal stability of dihydrofolate reductase (3). Although there was no significant stabilization observed in the pioneering work on a cyclic bovine pancreatic trypsin inhibitor prepared by chemical modification, several effects may have canceled out in this example (4).A recent development in protein chemistry, the use of selfsplicing proteins (often called inteins), has opened a general avenue to create a circular backbone topology as well as to ligate proteins and peptides in vitro. This procedure has been called expressed protein ligation or intein-mediated protein ligation (Refs. 5 and 6 and, for review, see Ref. 7). In this approach, an intein with an asparagine-to-alanine mutation in the active site is fused to one fusion partner. This mutation stops the enzymatic reaction at the stage of a C-terminal thioester of the fusion partner, selectively cleavable by thiols. The second peptide carrying an N-terminal cysteine acts as an S-nucleophile at the thioester group, forming a new peptide bond after S-N rearrangement. However, this approach has the disadvantage that it requires the nucleophilic thiol group of cysteine at the N terminus of one partner as well as a Cterminal thioester modification of the other partner, whose formation is catalyzed by the intein (5, 6). This reaction can be used to make cyclic peptides and proteins, but the intramolecular cyclization reaction always has to compete with other intermolecular reaction...