Thermal protein unfolding in photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in aqueous solution at pH 7.5 is studied by light attenuation and fluorescence measurements. The temporal behavior of protein unfolding (denaturation) is monitored by observation of spectral changes of the first absorption band of the flavin cofactor. The nPAC unfolding occurs irreversible in a bi-exponential manner (different melting time constants for proteins at nano-cluster surface and in nano-cluster interior). The nPAC apparent melting temperature (there half of the proteins are unfolded) is determined by light attenuation measurement (light scattering increases due to coalescing of unfolded protein nano-clusters) in the non-absorbing spectral region of the protein. A measurement standard is developed employing a staircase temperature heating and cooling profile. High thermal stability of nPAC nano-clusters in pH 7.5 aqueous solution was found with an apparent melting temperature of 60 °C.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Apparent melting temperature of 60 °C is determined by static light scattering measurement. ► Temporal development of protein unfolding is observed by flavin absorption spectra changes. ► Faster unfolding at surface and slower unfolding in interior of nPAC nano-clusters is observed. ► Enthalpy and entropy of irreversible protein unfolding are determined. ► Protein coalescing in denaturation process is quantified by light-scattering analysis.