The electron transfer flavoprotein (ETF) is a hub interacting with at least 11 mitochondrial flavoenzymes and linking them to the respiratory chain. Here we report the effect of the ETF alpha-T/I171 polymorphism on protein conformation and kinetic stability under thermal stress. Although variants have comparable thermodynamic stabilities, kinetically their behavior is rather distinct as ETF alpha-T171 displays increased susceptibility to cofactor flavin adenine dinucleotide (FAD) loss and enhanced kinetics of inactivation during thermal stress. Mimicking a fever episode yields substantial activity loss. However, the presence of substoichiometric concentrations of GroEL is sufficient to act as an effective buffer against long-term thermal denaturation. Our investigations are compatible with the notion that the ETF alpha-T171 variant displays an altered conformational landscape that results in reduced protein function under thermal stress. (C) 2011 Federation of European Biochemical Societies.