# Assessment of methods for evaluating structural stability of cell envelope fragments in hypersaline brines as biosignatures of ancient microbial life

## TL;DR

This paper evaluates methods to study how proteins and lipids from halophilic archaea remain stable in hypersaline environments, which is important for understanding ancient microbial life on Earth and Mars.

## Contribution

The study identifies technical challenges and potential solutions for analyzing biomolecular stability in hypersaline brines using various analytical techniques.

## Key findings

- NanoDSF is useful for analyzing multi-protein systems in hypersaline conditions.
- DSC may be applicable with new technology despite crystallization challenges.
- AUC has limited applicability due to viscosity-related artifacts in high-salinity environments.

## Abstract

The study of biomolecular stability of proteins and lipids in extreme saline environments is critical for understanding the preservation of potential microbial biosignatures of ancient life on Earth and other planetary bodies, including Mars. In this study, we evaluate the compatibility of several analytical techniques, Nano-Differential Scanning Fluorometry (NanoDSF), Analytical Ultracentrifugation (AUC), and Differential Scanning Calorimetry (DSC) with hypersaline brine analogues of Early Earth and Early Mars conditions. Using the halophilic archaeon Halobacterium salinarum as a model, we examine the structural stability of proteins within cell envelope fragments from dead cells, focusing on their preservation potential in complex brines. The results reveal significant technical challenges in studying macromolecules in high-salinity environments, including crystallisation during DSC and NanoDSF, viscosity-related artifacts in AUC, and reduced fluorescence signals in NanoDSF due to the low tryptophan content of membrane proteins from halophilic archaea. Nevertheless, NanoDSF proved useful for analysing multi-protein systems and DSC may be applicable using new generation technology, while AUC showed limited applicability under extreme saline conditions. These findings provide crucial insights into the methodologies for studying the stability of halophilic biomolecules in brine environments and the limitations of current techniques in extreme settings.

The online version contains supplementary material available at 10.1038/s41598-025-11211-7.