A thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.

La température est un paramètre crucial dans le fonctionnement du monde vivant, notamment de la machinerie moléculaire (les protéines) dont la stabilité et l’activité en dépendent sensiblement. Celles-ci sont souvent considérées comme étant équivalentes : si une protéine fonctionne, c’est qu’elle est stable, et vice-versa. Cependant, les protéines des organismes thermophiles, qui prolifèrent dans de températures élevées, sont stables à température ambiante, mais y présentent une faible activité. Cette dernière est optimale à la température de croissance de l’organisme hôte. Lorsqu’on parle de stabilité et d’activité protéique, la rigidité mécanique est souvent utilisée comme paramètre pertinent, offrant une explication simple et attractive à la fois pour la stabilité thermodynamique à haute température et au manque d’activité à des températures plus modérés. La réalité s’avère souvent plus complexe, et les mécanismes moléculaires reliant rigidité/flexibilité avec la stabilité et l’activité sont encore mal compris. Dans ce travail, nous abordons le problème au travers de trois systèmes. Nous avons examiné l’activation thermique des modes fonctionnels du domaine G de la protéine EF ainsi que les homologues mésophiles et thermophiles de la déshydrogénase Lactate/Malate. Par ailleurs, nous avons mis en évidence l’existence d’un paramètre unique (la moyenne des fluctuations atomiques) permettant d’expliquer la dynamique de la protéine lysozyme près de son point de fusion, et ce quelle que soit la nature de l’environnement autour de la protéine (qui décale le point de fusion). Nos conclusions se basent principalement sur une approche in silico où la dynamique moléculaire et des techniques d’échantillonnage améliorées sont utilisées et sont complémentées par des expériences de diffraction de neutrons / Temperature is one of the major factors governing life as demonstrated by the fine tuning of stability and activity of the molecular machinery, proteins in particular. The structural stability and activity of proteins have been often presented as equivalent. However, the thermophilic proteins are stable at ambient condition, but lack activity, the latter recovered only when the temperature increases to match that of the optimal growth condition for the hosting organism. In discussing the protein stability and activity, mechanical rigidity is often used as a relevant parameter, offering a simple and appealing explanation of both the extreme thermodynamic stability and the lack of activity at low temperature. The reality, however, illustrates the complexity of the rigidity/flexibility trade off in ensuring stability and activity through intricate thermodynamic and molecular mechanisms. Here we investigate the problem by studying three study cases. These are used to relate the thermal effects on mechanical properties and the stability and activity of the proteins. For instance, we have probed the thermal activation of functional modes in EF G-domain and Lactate/Malate dehydrogenase mesophilic and thermophilic homologues and verified a “universal” scaling of atomistic fluctuation of the Lysozyme approaching the melting in different environmental conditions. Our conclusions largely rest on an in silico approach, where molecular dynamics and enhanced sampling techniques are utilized, and are often complemented with neutron scattering experiments