Thermodynamic assessment of domain‐domain interactions and in vitro activities of mesophilic and thermophilic ribosome recycling factors

Abstract

Ribosome recycling factor (RRF) is essential for bacterial growth. Structural studies revealed that RRF consists of two domains connected by two short polypeptides at the hinge region. Here, we evaluated the intrinsic stabilities (ΔG*s) of the two domains and the free energy of the domain-domain interactions (ΔG(int)) for mesophilic RRF (RRF from Escherichia coli, EcRRF) and thermophilic RRF (RRF from Thermus thermophilus, TtRRF) by using differential scanning calorimetry and circular dichroic spectroscopy. Despite single endothermic peaks, a higher than unity value for the ratio of calorimetric enthalpy to van't Hoff enthalpy of the unfolding indicated the presence of unfolding intermediates for both RRFs. Deconvolution analysis based on statistical thermodynamics employing multiple states of the unfolding process with domain-domain interactions allowed us to determine ΔG*s of each domain and ΔG(int). The results indicated that domain I has a higher unfolding temperature than domain II and that it stabilizes domain II through ΔG(int), giving rise to an apparent single peak of unfolding. Interestingly, the estimated ΔG(int) values of 6.28 kJ/mol for EcRRF and 26.28 kJ/mol for TtRRF reflect the observation that only EcRRF has recycling activity at ambient temperature. Our present study suggests the importance of a moderate ΔG(int) for biological activity of multidomain proteins.