Thermal stability of chemically denatured green fluorescent protein (GFP): A preliminary study

Abstract Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria . The protein consist of 238 amino acids and produces green fluorescent light ( λ max =508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide –Ser 65 –Tyr 66 –Gly 67 –, which buried into β-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1–6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 °C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6–0.2 kJ mol −1 ), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol −1 . First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in β-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.