A new record: The largest chemically synthesized polypeptide composed of 304 residues, which corresponds to folded K48‐tetraubiquitin, has been achieved (see scheme). The presented synthetic method could be applied to any of the remaining tetraubiquitin chains, and should ultimately assist ongoing efforts to unravel how the remarkable diversity of ubiquitin signaling is achieved.

The magnetosome, a biomineralizing organelle within magnetotactic bacteria, allows their navigation along geomagnetic fields. Magnetosomes are membrane-bound compartments containing magnetic nanoparticles and organized into a chain within the cell, the assembly and biomineralization of magnetosomes are controlled by magnetosome-associated proteins. Here, we describe the crystal structures of the magnetosome-associated protein, MamA, from Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. MamA folds as a sequential tetra-trico-peptide repeat (TPR) protein with a unique hook-like shape. Analysis of the MamA structures indicates two distinct domains that can undergo conformational changes. Furthermore, structural analysis of seven crystal forms verified that the core of MamA is not affected by crystallization conditions and identified three protein–protein interaction sites, namely a concave site, a convex site, and a putative TPR repeat. Additionally, relying on transmission electron microscopy and size exclusion chromatography, we show that highly stable complexes form upon MamA homooligomerization. Disruption of the MamA putative TPR motif or N-terminal domain led to protein mislocalization in vivo and prevented MamA oligomerization in vitro. We, therefore, propose that MamA self-assembles through its putative TPR motif and its concave site to create a large homooligomeric scaffold which can interact with other magnetosome-associated proteins via the MamA convex site. We discuss the structural basis for TPR homooligomerization that allows the proper function of a prokaryotic organelle.

The combination of native chemical ligation and desulfurization is considered a powerful strategy in protein synthesis. Homogeneous desulfurization conditions based on a radical induced reaction have been widely used in the syntheses of various challenging proteins and their analogues. However, the presence of aryl thiols in the reaction mixture as a ligation catalyst hampers one-pot ligation/ desulfurization, hence mandating additional purification/lyophilization steps prior to desulfurization. This significantly reduces the yield and prolongs the ligation process. Here we report that the use of preformed peptide-aryl thioester allows for efficient one-pot ligation and desulfurization. This approach was tested successfully for various model peptides including the synthesis of ubiquitin from two fragments. However, in the case of the synthesis of di-ubiquitin chains, where the ligation is mediated by d-mercaptolysine to form an isopeptide bond, excess aryl thiol was required for efficient ligation, necessitating purification prior to desulfurization. To overcome these obstacles, we found that functionalization of the aryl thiol with a hydrazide moiety enabled, after the ligation step, its capture by resin-aldehyde to permit direct desulfurization. Altogether, these approaches should facilitate protein synthesis with improved efficiency in yields and time.

Neuer Rekord: Das größte chemisch synthetisierte Polypeptid besteht aus 304 Resten und entspricht gefaltetem K48‐Tetraubiquitin (siehe Schema). Da die Synthesemethode auch auf die übrigen Tetraubiquitinketten angewendet werden kann, könnte sie auf lange Sicht dabei helfen, die bemerkenswerte Vielfalt der Ubiquitin‐Signalwege aufzuklären.

The HIV‐1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV‐1 lifecycle, thus making Rev an attractive target for the design of anti‐HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self‐assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one‐pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.