Local and Coupled Thermodynamic Stability of the Two-Domain and Bifunctional Enzyme SlyD from Escherichia coli

Abstract

SlyD (sensitive to lysis D) is a protein folding helper enzyme comprising peptidylprolyl isomerase as well as chaperone activities at the respective FKBP and IF domains. Both domains coact concerning the peptidylprolyl isomerase activity on protein substrates. Using various biophysical techniques and NMR spectroscopy, the local and global thermodynamic stability of the variant (1-165) of SlyD from Escherichia coli (SlyD*) was characterized. Structurally, both domains are rather independent. The urea-induced unfolding transitions of the two domain protein monitored by 2D NMR spectroscopy and amide proton exchange experiments, however, showed that the IF domain experiences a reduced local stability under both native and unfolding conditions compared to the FKBP domain. Nevertheless, the entire protein shows highly cooperative unfolding at elevated denaturing conditions. This cooperativity is significantly reduced in a SlyD* variant missing the IF domain. The quite low local stability due to high internal fluctuations of the IF domain might be the prerequisite for the ubiquitous chaperone function of SlyD. One physiological role of the metallochaperone SlyD is divalent cations binding. Nickel binds only to the FKBP domain but extensively increases the thermodynamic stability of both SlyD domains, verifying the coupled stability of the domains.