Incorporation of a Mo in a Rubredoxin-Type Centre of a de Novo Designed α3-DIV-L21C Three Helix Bundle Peptide

• Autor(a) principal: Bragança, Pedro Miguel da Silva
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/89462
• topic: a de novo protein design; molybdenum; DMSOR family; rubredoxin; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Approximately one-third of all proteins and enzymes require one or more metal atoms to perform their catalytic functions. Presently, the rational design and engineer of small, simple and stable peptides scaffolds to mimic catalytic metal-centres of complex proteins is an important goal in the area of protein design and search for centres with environmental, biotechnological and basic research impact. In this work, a de novo designed α3DIV-L21C peptide that possesses a rubredoxin-type centre as the metal-binding site was used to incorporate a Mo atom to mimic, specifically, the tetrathiolate environment found in the active-site of molybdenum-bis pyranopterin guanosine dinucleotide-containing enzyme (Mo-bis PGD). The Mo-α3DIV-L21C peptide reconstitution was acceded by ICP-AES to optimize the reconstitution conditions tested and a 1:1 peptide/metal ratio was obtained using a 1:10 peptide/metal ratio for reconstitution. Using UV-vis spectroscopy, the emergence of two broad bands, with a maximum absorption between 310-330 nm (ε320nm = 7400 M-1.cm-1) and 460-475 nm (ε470nm = 2000 M-1.cm-1), confirm the metal incorporation within the tetracysteinyl environment. Circular dichroism (CD) studies, in the visible region, further confirmed the metal incorporation through the appearance of a positive band with a maximum at 394 nm. Furthermore, secondary structure profile and tertiary structure fingerprint were examined, under TCEP-induced reducing and non-reducing conditions, as well as thermal stability studies in both far-UV and visible regions. Differential Scanning Calorimetry (DSC) was used to perform a thermal denaturation analysis and define thermodynamic parameters for Mo-α3DIVL21C peptide. Thermogram fitted well to a reversible monomeric two-state model, with a Tm value of 66 ºC, a ΔHcal value of 846.4 kJ.mol-1 and a ΔHvH value of 920.03 kJ.mol-1. Preliminary electrochemical results through the use of cyclic voltammetry (in a thin-layer regime) suggest an initial oxidation state of Mo(VI), within the metal-binding site, and formal potential (E0’) of -406 mV vs. NHE with a capability to undergo one-electron transfer process (Mo(VI)/Mo(V)). Moreover, reducing tests attained with different reducing agents and followed by spectroscopy seems in agreement with this assumption.