biology Article The Effects of Temperature and Pressure on Protein-Ligand Binding in the Presence of Mars-Relevant Salts † Nisrine Jahmidi-Azizi 1 , Rosario Oliva 1, *, Stewart Gault 2 , Charles S. Cockell 2 and Roland Winter 1, *   Citation: Jahmidi-Azizi, N.; Oliva, R.; Gault, S.; Cockell, C.S.; Winter, R. The Effects of Temperature and Pressure on Protein-Ligand Binding in the Presence of Mars-Relevant Salts. Biology 2021, 10, 687. https:// doi.org/10.3390/biology10070687 Academic Editors: Dmitri Davydov and Christiane Jung Received: 25 June 2021 Accepted: 16 July 2021 Published: 20 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Physical Chemistry I—Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany; nisrine.jahmidi@tu-dortmund.de 2 UK Centre for Astrobiology, SUPA School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, UK; s.a.gault@sms.ed.ac.uk (S.G.); c.s.cockell@ed.ac.uk (C.S.C.) * Correspondence: rosario.oliva@tu-dortmund.de (R.O.); roland.winter@tu-dortmund.de (R.W.) † The manuscript is dedicated to Dr. Gaston Hui Bon Hoa. Simple Summary: Interactions of ligands with proteins are central to all reactions in the biological cell. How such reactions are affected by harsh environmental conditions, such as low temperatures, high pressures, and high concentrations of biologically destructive salts, is still largely unknown. Our work focused on specific salts found on Mars to understand whether the planet’s potentially liquid, water-rich subsurface harbors conditions that are theoretically favorable for life. Our data show that, while magnesium chloride and sulfate do not significantly alter protein–ligand interactions, the perchlorate ion strongly affects protein–ligand binding. However, the temperature and pressure conditions encountered on Mars do not necessarily preclude protein–ligand interactions of the type studied here. Abstract: Protein–ligand interactions are fundamental to all biochemical processes. Generally, these processes are studied at ambient temperature and pressure conditions. We investigated the binding of the small ligand 8-anilinonaphthalene-1-sulfonic acid (ANS) to the multifunctional protein bovine serum albumin (BSA) at ambient and low temperatures and at high pressure conditions, in the presence of ions associated with the surface and subsurface of Mars, including the chaotropic perchlorate ion. We found that salts such as magnesium chloride and sulfate only slightly affect the protein–ligand complex formation. In contrast, magnesium perchlorate strongly affects the interaction between ANS and BSA at the single site level, leading to a change in stoichiometry and strength of ligand binding. Interestingly, both a decrease in temperature and an increase in pressure favor the ligand binding process, resulting in a negative change in protein–ligand binding volume. This suggests that biochemical reactions that are fundamental for the regulation of biological processes are theoretically possible outside standard temperature and pressure conditions, such as in the harsh conditions of the Martian subsurface. Keywords: protein–ligand binding; high pressure; Martian salts; perchlorate; BSA; ANS 1. Introduction Protein–ligand recognition and binding are fundamental to all biochemical processes and are essential for all life forms [1–5]. Hence, elucidating the nature and strength of the driving forces involved in the ligand binding processes is of particular interest in the biosciences. In most cases, non-covalent bonds, such as electrostatic and hydrophobic interactions, ensure formation of the protein–ligand complexes [5]. In a molecular picture, protein–ligand interactions may not strictly follow a simple binding process; instead, they may be accompanied by conformational as well as hydration changes of the protein and potentially also the ligand. Owing to the inherent complexity of the process, many aspects of ligand binding have not been fully explored, yet. This is particularly true for Biology 2021, 10, 687. https://doi.org/10.3390/biology10070687 https://www.mdpi.com/journal/biology