Laboratorio Of Biofisica Molecular

Ribeirão Preto, Brazil

Laboratorio Of Biofisica Molecular

Ribeirão Preto, Brazil
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Mendes L.F.S.,Laboratorio Of Biofisica Molecular | Garcia A.F.,Laboratorio Of Biofisica Molecular | Kumagai P.S.,University of Sao Paulo | De Morais F.R.,São Paulo State University | And 5 more authors.
Scientific Reports | Year: 2016

Among all proteins localized in the Golgi apparatus, a two-PDZ (PSD95/DlgA/Zo-1) domain protein plays an important role in the assembly of the cisternae. This Golgi Reassembly and Stacking Protein (GRASP) has puzzled researchers due to its large array of functions and relevance in Golgi functionality. We report here a biochemical and biophysical study of the GRASP55/65 homologue in Cryptococcus neoformans (CnGRASP). Bioinformatic analysis, static fluorescence and circular dichroism spectroscopies, calorimetry, small angle X-ray scattering, solution nuclear magnetic resonance, size exclusion chromatography and proteolysis assays were used to unravel structural features of the full-length CnGRASP. We detected the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. Our results indicate an unusual behavior of CnGRASP in solution, closely resembling a class of intrinsically disordered proteins called molten globule proteins. To the best of our knowledge, this is the first structural characterization of a full-length GRASP and observation of a molten globule-like behavior in the GRASP family. The possible implications of this and how it could explain the multiple facets of this intriguing class of proteins are discussed.


Barroso R.P.,Laboratorio Of Biofisica Molecular | Basso L.G.M.,Laboratorio Of Biofisica Molecular | Costa-Filho A.J.,Laboratorio Of Biofisica Molecular
Chemistry and Physics of Lipids | Year: 2015

A detailed molecular description of the mechanism of action of the antimalarial drug amodiaquine (AQ) is still an open issue. To gain further insights on that, we studied the interactions of AQ with lipid model membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) by spin labeling electron spin resonance (ESR) and differential scanning calorimetry (DSC). Both techniques indicate a coexistence of an ordered DPPS-rich domain with a disordered DPPC-rich domain in the binary DPPC/DPPS system. We found that AQ slightly lowered the melting transition temperatures associated to both domains and significantly increased the enthalpy change of the whole DPPC/DPPS phase transition. DSC and ESR data also suggest that AQ increases the number of DPPC molecules in the DPPC-rich domains. AQ also causes opposing ordering effects on different regions of the bilayer: while the drug increases the ordering of the lipid acyl chains from carbon 7 to 16, it decreases the order parameter of the lipid head group and of carbon 5. The gel phase was mostly affected by the presence of AQ, suggesting that AQ is able to influence more organized lipid domains. Moreover, the effects of AQ and cholesterol on lipid acyl chain ordering and mobility were compared at physiological temperature and, in a general way, they are similar. Our results suggest that the quinoline ring of AQ is located completely inside the lipid bilayers with its phenol ring and the tertiary amine directed towards the head group region. The nonspecific interaction between AQ and DPPC/DPPS bilayers is a combination of electrostatic and hydrophobic interactions. © 2014 Elsevier Ireland Ltd.

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