The small heat shock protein (smHsp) Lo18 from lactic acid bacteria Oenococcus oeni reduces in vitro thermal aggregation of proteins and modulates the membrane fluidity of native liposomes. An absence of information relating to the way in which the smHsp demonstrates a stabilizing effect for both proteins and membranes prompted this study. We expressed three Lo18 proteins with amino acid substitutions in Escherichia coli to investigate their ability to prevent E. coli protein aggregation and their capacity to stabilize E. coli whole-cell membranes. Our results showed that the alanine 123 to serine substitution induces a decrease in chaperone activity in denaturated proteins, and that the tyrosine 107 is required for membrane stabilization. Moreover, this study revealed that the oligomeric structures of proteins with amino acid substitutions do not appear to be modified. Our data strongly suggest that different amino acids are involved in the thermostabilization of proteins and in membrane fluidity regulation and are localized in the alpha-crystallin domain.