Polymerization behavior of a bifunctional ubiquitin monomer as a function of the nucleophile site and folding conditions.

Biopolymers with repeating modules composed of either folded peptides or tertiary protein domains are considered some of the basic biomaterials that nature has evolved to optimize for energy efficient synthesis and unique functions. Such biomaterials continue to inspire scientists to mimic their exceptional properties and the ways that nature adopts to prepare them. Ubiquitin chains represent another example of nature's approach to use a protein-repeating module to prepare functionally important biopolymers. In the current work, we utilize a novel synthetic strategy to prepare bifunctional ubiquitin monomers having a C-terminal thioester and a nucleophilic 1,2-aminothiol at a desired position to examine their polymerization products under different conditions. Our study reveals that such analogues, when subjected to polymerization conditions under different folding states, afford distinct patterns of polymerization products where both the dynamic and the tertiary structures of the chains play important roles in such processes. Moreover, we also show that the presence of a specific ubiquitin-binding domain, which binds specifically to some of these chains, could interfere selectively with the polymerization outcome. Our study represents the first example of examining the polymerization of designed and synthetic repeating modules based on tertiary protein domains and affords early lessons in the design and synthesis of biomaterial. In regards to the ubiquitin system, our study may have implications on the ease of synthesis of ubiquitin chains with varying lengths and types for structural and functional analyses. Importantly, such an approach could also assist in understanding the enzymatic machinery and the factors controlling the assembly of these chains with a desired length.

Self-recognition mechanism of MamA, a magnetosome-associated TPR-containing protein, promotes complex assembly.

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.

Chemical synthesis and expression of the HIV-1 Rev protein.

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.