Ultra-high resolution structure determination of transition metal substituted human carbonic anhydrase 2 – inhibitor complexes

Paramagnetic Nuclear Magnetic Resonance (NMR) is developing to aid the characterization of paramagnetic molecules, whose paramagnetic centers changes the spectroscopic proprieties of said molecules. These paramagnetic centers can be exploited to overcome some troublesome aspects of NMR, such as sensitivity, by increasing the number of experiments. To aid this development, we used human Carbonic Anhydrase II (hCAII), which is a model protein that contains zinc(II) in its active center, which is diamagnetic. hCAII is an enzyme capable of interconverting carbon dioxide to bicarbonate, making it one of the most important proteins in life. Several comprehensive studies have structurally and functionally characterized hCAII making it an excellent model protein. Furthermore, the metal ion in the active center can be substituted by other transition metals ions (see chapter 1), such as cobalt(II) (see chapter 3), nickel(II) (see chapter 4) and copper(II) (see chapter 5), which are paramagnetic that will help answering different problems described in this thesis. The ion cobalt(II), explored in chapter 3, can induce considerable changes on the NMR observables and is useful to understand the interactions of ligands with proteins. For this we used cobalt(II)-hCAII and used NMR, Electron Paramagnetic Resonance (EPR) and X-ray crystallography to characterize the interaction of thiocyanate under high concentrations with the hCAII. The addition of 500 mM of sodium thiocyanate changes the dynamics of the protein without changing the protein structure. Solid-state NMR (SSNMR) is another field of NMR where the methodological and practical aspects are currently under development to reach better sensitivity and resolution. We proposed the usage of nickel(II)-hCAII as a paramagnetic molecule to increase the amount of tools in SSNMR, which is explored in chapter 4. The nickel(II) ion is capable of breaking the dipolar bath by changing the frequency of the nuclei close to the paramagnetic center, thus increasing the resolution and sensitivity of the SSNMR experiments. Furthermore, in parallel we discovered that hCAII is capable of binding two nickel(II) ions, one in the active center, as expected and described in literature, and one in the N-terminal site of the protein, a novel discovery. The description of the paramagnetic effects, such as the Pseudocontact Shifts (PCS), in the NMR observables have been subjected to debate, where different treatments of theoretical equations were clashing. The experimental proof to determine which equation holds true is fully described in chapter 5. For this, we developed copper(II)-hCAII to acquire NMR and EPR data under the same conditions, and determine which equations describe better the PCS. The data interpretation from different techniques (both NMR and EPR) led us to conclude that the original treatment from Kurland and McGarvey equation is the correct one.