Biology subjects

Naganathan, A. N.

Publications and source records attributed to Naganathan, A. N..

2 recordsLinked to original sources

bioRxiv2021

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

biophysics↗

bioRxiv2020

The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS Model

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, ALS. Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a co-factor derived membrane association model wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association mediated toxic aggregation and survival time scale after ALS diagnosis.

biophysics↗