Effect of ultrasound on the function and structure of a membrane protein: The case study of photosynthetic Reaction Center from Rhodobacter sphaeroides

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30 W and 20 kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32 min in detergent micelles, ranged from 3.8 to 6.5 min in the different proteoliposomes formulations, and 5.5 min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to, the sonication-induced denaturation, in agreement with the scattering increase observed in solution.