Diverse crystalline protein scaffolds through metal-dependent polymorphism.

As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co-crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein-metal-organic frameworks (POFs). In addition, metal-dependent polymorphic crystals allow direct comparison of metal coordination preferences.

Wave fetch and distance from the ocean determine the distribution of macroplastics in the intertidal zone of central Spitsbergen, Arctic.

In this study, we estimated the variety and distribution of macroplastics in the central part of Spitsbergen, Svalbard archipelago, Arctic. All marine litter photos were georeferenced, then identified using the OSPAR (2010) classification guide. The majority (90% of all objects) of marine debris was macroplastic with average number in the study area being 2.0 ± 0.4 objects per 100 m. It was determined that the full variety of macroplastic categories in the study area can be found after surveying approx. 8 km of coastline. Correlation analysis showed that the amount of macroplastic accumulated on the beaches decreases with distance from the open ocean and increases with wave fetch. When zoning the entire study area on the basis of a cluster analysis of the distribution of macroplastics, it was found that the geographical proximity of the sections is less important than the wave fetch.

Easy Production of "Difficult Peptides" Using Cell-Free Protein Synthesis and a New Methionine Analogue as a Latent Peptide Cleavage Site.

Aliphatic γ-chloro-α-amino acids incorporated in place of their canonical analogues through cell-free protein synthesis act as heat-labile linkers, offering a useful strategy for the straightforward production of target peptides as fusion proteins, from which the targets are readily released. Until now, the natural abundance of aliphatic amino acids in peptides has limited the scope of the method, as it leads to undesired cleavage sites in synthesized products, but here the authors report the development of a new cleavable chloro amino acid that incorporates in place of the relatively rare amino acid methionine, thus greatly expanding the scope of producible targets. This new strategy is employed for simplified peptide synthesis with a methionine-free fusion partner, allowing single-site incorporation of the cleavable linker for clean release and easy purification of the target peptide. Its utility is demonstrated through the straightforward preparation of two peptides reported to be challenging targets and not accessible through standard solid-phase chemical methodologies, as well as analogues.

Immobilization of Enzymes in Protein Films.

Heterogeneous biocatalysis usually involves the use of immobilized enzymes on solid supports. Enzymes have suitable properties in terms of efficiency and selectivity for use as immobilized catalysts. Different approaches have been developed for effective immobilization, including adsorption, covalent binding, entrapment, encapsulation, and cross-linking. Those systems offer some advantages with regard to homogeneous catalysts in solution, such as low costs, easy separation and recovery of the catalyst, reusability, and enzymatic stability. Here, we describe a new approach for the immobilization of active enzymes into homogenous films composed solely of scaffolding proteins that differs from the standard methods of enzyme immobilization on solid supports.

Protein-directed crystalline 2D fullerene assemblies.

Water soluble 2D crystalline monolayers of fullerenes grow on planar assemblies of engineered consensus tetratricopeptide repeat proteins. Designed fullerene-coordinating tyrosine clamps on the protein introduce specific fullerene binding sites, which facilitate fullerene nucleation. Through reciprocal interactions between the components, the hybrid material assembles into two-dimensional 2 nm thick structures with crystalline order, that conduct photo-generated charges. Thus, the protein-fullerene hybrid material is a demonstration of the developments toward functional materials with protein-based precision control of functional elements.

Repeat proteins as versatile scaffolds for arrays of redox-active FeS clusters.

Arrays of one, two and four electron-transfer active [4Fe-4S] clusters were constructed on modular tetratricopeptide repeat protein scaffolds, with the number of clusters determined solely by the size of the scaffold. The constructs show reversible redox activity and transient charge stabilization necessary to facilitate charge transfer.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

In situ deprotection and incorporation of unnatural amino acids during cell-free protein synthesis.

The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; α-amino form-, acet-, trifluoroacet-, and benzamides; and side-chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5-dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoro- and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.