Stabilization of a prokaryotic LAT transporter by random mutagenesis

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.

A non-sovereign modernity: attempts to engineer stability in the Balkans 1820–90

Social theory almost invariably equates modernity with the sovereign state. This equation must be nuanced because the modern era and modern strategies of international stability have contained non-sovereign units. In the nineteenth century, the Great Powers tried to create international stability by engineering forms of rule in Europe. These strategies built on distinctively modern ideas: the possibility of radically breaking with the past, redesigning political organisations, and actively controlling political events through rational planning. Throughout the century the Great Powers alternated between creating non-sovereign units and creating sovereign units as instruments in these stabilising strategies. The degree of trust between the Great Powers accounts for the shift between the two strategies: they tended to create non-sovereign units when mutual trust was high and sovereign ones when trust was low. This article analyses Great Power strategies of designing forms of rule in the Balkans between 1820 and 1878. Like in previous centuries, nineteenth-century Europe actually consisted of two parallel but connected systems: the egalitarian system of sovereign states and a system of non-sovereign entities. Non-sovereign units disappeared only late in the century and this process was affected by the increasing rivalry and mistrust between the sovereign states.

Reflections on the Great Recession of 2008–09

In the years that preceded the Great Recession of 2008–09, many financial institutions in the developed world engaged in practices that increased the risk of systemic failure in the markets. The economics profession itself, overly confident of its ability to engineer stability, encouraged financial actors to exceed the traditional dictates of caution. The financial crisis that ensued also revealed many fault lines. One of the most glaring was an absence of international coordination, which encouraged regulatory shopping. Dramatic actions taken by public authorities in the fall of 2008 avoided collapse, but left a legacy of increased moral hazard, to which the best long-term answer is radical structural reform. Asian emerging markets were, by and large, spared the worst of the storm. They should learn from the mistakes of their peers in the developed world, but not reject wholesale the benefits of free markets.