A carregar...
Projeto de investigação
Ultra - High resolution structure determination of cobalt substituted human carbonic anhydrase 2 - Inhibitor complexes
Financiador
Autores
Publicações
Elucidating the concentration-dependent effects of thiocyanate binding to carbonic anhydrase
Publication . Silva, José Malanho; Cerofolini, Linda; Carvalho, Ana Luísa; Ravera, Enrico; Fragai, Marco; Parigi, Giacomo; Macedo, Anjos L.; Geraldes, Carlos F. G. C.; Luchinat, Claudio; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier
Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.
Ultra-high resolution structure determination of transition metal substituted human carbonic anhydrase 2 - inhibitor complexes
Publication . Silva, José Pedro Malanho da; Macedo, Maria dos Anjos; Geraldes, Carlos
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.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
OE
Número da atribuição
PD/BD/135180/2017