Experimental study of proteome halophilicity using nanoDSF: a proof of concept

Abstract

40Adaption to environmental conditions is reflected by protein adaptation. In particular, 41 proteins of extremophiles display distinctive traits ensuring functional, structural and 42 dynamical properties under permanently extreme physical and chemical conditions. While it 43 has mostly been studied with approaches focusing on specific proteins, biophysical 44 approaches have also confirmed this link between environmental and protein adaptation at 45 the more complex and diverse scale of the proteome. However, studies of this type remain 46 challenging and often require large amounts of biological material. 47We report here the use of nanoDSF as a tool to study proteome stability and solubility 48 in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results 49 show that, as with single halophilic protein studies, proteome stability was correlated to the 50 concentration of NaCl or KCl under which the cells were lysed and hence the proteome 51 exposed. This work highlights that adaptation to environmental conditions can be 52 experimentally observed at the scale of the proteome. Still, we show that the biochemical 53 properties of single halophilic proteins can only be partially extrapolated to the whole 54 proteome. 55Page 2 of 23 Extremophiles 9 310 observed between H. marismortui proteome and MDH. Nonetheless, this stresses that the 311 behaviour of the proteome can only partially be extrapolated from that of a given enzyme. 312Our experimental setup allowed little to no observation of peaks of first derivative of 313 ratio of 350/330nm fluorescence during the cooling step following the heating. When 314 observed, these renaturation peaks were less distinctive than those associated with 315 denaturation. Moreover, no peak of first derivative of light scattering could be observed at all, 316 showing that aggregation process was also mostly irreversible. Since aggregation peaks 317 happened after denaturation peaks during the heating step, irreversible aggregation may be 318 the reason why renaturation was very limited, even with the pure MDH. In the case of the 319 proteome, the limited renaturation could be associated with a specific subset of proteins 320 whose denaturation was reversible, rather than a partial renaturation of the whole proteome. 321 This was unlike the ß-lactamase from the moderately halophilic bacterium Chromohalobacter 322 sp. 560 which has been shown to refold spontaneously after thermal denaturation in 0.2M 323 NaCl (Tokunaga et al. 2004). However, after being expressed in non-halophilic Escherichia coli 324 cells where it has undergone low salt denaturation, H. marismortui MDH can be refolded in 325 vitro by increasing the salinity (Cendrin et al. 1993;Franzetti et al. 2001). Similarly, a 326 nucleoside diphosphate kinase from the extremely halophilic archaea H. salinarum, which has 327 been expressed in E. coli, has also been shown to require high salt concentration for refolding 328 while being stable and active in low salinity conditions (Ishibashi et...

Effects of chaotropic salts on global proteome stability in halophilic archaea: Implications for life signatures on Mars

“…To enable statistical comparisons, experiments with KCl or NaCl only and with salt combinations (except KCl‐CaCl 2 ) were independently replicated for the five strains: three additional experimental points with fewer salt concentrations for KCl or NaCl only and two additional experimental points with the same conditions for KCl‐MgCl 2 and NaCl‐(per)chlorate combinations. The temperatures associated with the peak value of the first derivative of ratio of 350/330 nm fluorescence associated with the main dominant denaturation events, which we previously defined as proteome melting temperatures ( T Pm ) (Carré et al, 2021), were retrieved. The slope of lines fitting T Pm as a function of the concentration of the salt were also calculated.…”

“…With this respect, nanoDSF is a valuable approach to probe molecular response to chemical conditions at the proteome scale. In the present work, we extended our proof of concept experiment (Carré et al, 2021) to the comparison of several halophilic extremophiles. Obviously, the intrinsic fluorescence signal depends on the existence of aromatic residues within the sample and comparison between strains may be biased if aromatic residue content differed.…”

“…To investigate proteome responses to KCl, NaCl, MgCl 2 , CaCl 2 and to combinations between them, we first used lysates of cells of H. marismortui in exponential phase. After dilution in various salt concentrations to reach the final desired composition and centrifugation, the supernatants, which contained the soluble fraction of the proteomes, were analysed using nanoDSF to derive proteome thermal denaturation and thermal aggregation curves as described in Carré et al (2021). Untreated thermal aggregation and denaturation signals in various salt conditions are presented in supplementary material section (Figures S2-S5).…”

“…By using label‐free differential scanning fluorimetry (nanoDSF), a method allowing analysis of thermal denaturation and thermal aggregation of proteins, we have recently revealed a difference between the stabilizing effect of KCl and NaCl on the proteome of the halophilic archaeon Haloarcula marismortui (Carré et al, 2021). In this study, we used this novel approach to explore the effects of combinations between kosmotropic (KCl, NaCl) and chaotropic salts (MgCl 2 , CaCl 2 , NaClO 3 , NaClO 4 , Mg(ClO 4 ) 2 and Ca(ClO 4 ) 2 ) on proteome stability in five different strains of Haloarchaea , including Mg‐tolerant strains.…”