Author(s):
Carvalho, Sofia Baptista de 
Date: 2011
Persistent ID: http://hdl.handle.net/10451/8475
Origin: Repositório da Universidade de Lisboa
Subject(s): S100B; Folding de proteínas; Iões metálicos; Dicroísmo circular; Estabilidade térmica; Alterações conformacionais; Teses de mestrado - 2011
Description
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011 S100B is a Ca2+, Cu2+ and Zn2+-binding protein highly expressed in human brain, with a intra and extracellular function. It is involved in several pathological processes in which metal homeostasis is imbalanced, such as AD and ALS, which account for a modified protein function. Metal binding to S100B induces conformational changes. However, the mechanisms underlying this modulation and how it affects protein stability remain uncharacterized. Here, we extensively address the effects of metal binding on the folding and stability of S100B. The physiological environment in which S100B accumulates, such as the synaptic cleft, is metal ion rich so the protein{metal interplay is relevant to understand S100B physiological (and pathophysiological) role. With this purpose we have studied the native form of human S100B as well as a Cys-to-Ser mutated variant, S100B Δ Cys, which abolishes metal bonding. Our results showed that Cu2+ and Zn2+-binding is responsible for substantial modifications in protein conformation affording secondary structure changes and promoting aggregation. Thermal unfolding experiments indicated that Cu2+ and Zn2+ have the ability to destabilize the protein (apo-no aggregation, Cu2+-Tagg=68ºC, Zn2+-Tagg=65ºC)leading to a total loss of secondary structure and protein aggregation. Ca2+-binding does not have a large effect on S100B conformation and stability, yielding a mixture of α-helixand β-sheet secondary structure after denaturation, without forming aggregates. The study of Cu2+ and Zn2+ binding competition revealed that Cu2+ has the ability to displaceZn2+ from the binding site, with a destabilizing effect (ΔTagg = 35ºC). However,for S100B ΔCys, although Cu2+ also displaces Zn2+, it has the opposite effect stabilizing the protein (ΔTagg = - 5ºC). We observed that Cu2+, but not Zn2+ or Ca2+, has the ability to promote intermolecular disulfide bond formation between S100B subunits. We hypothesize that metal induced conformational changes may account for the buildup of conformers that have increased oligomerization propensity and that this may play a role in S100B function and on its interactors. This possibility is particularly relevant considering the interplay of S100B with the Amyloid-β peptide and with Receptor for Advanced Glycation EndProducts, which are, all together, suggestive of an even more interesting engagement of S100B in Alzheimer's Disease.
Document Type
Master Thesis
Language
English
Advisor(s)
Gomes, Cláudio M.; Coutinho, Ana Isabel Abrantes, 1965-
