Diogo Vila-Viçosa

Reversibility of prion misfolding: insights from constant-pH molecular dynamics simulations.

The prion protein (PrP) is the cause of a group of diseases known as transmissible spongiform encephalopathies (TSEs). Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are examples of TSEs. Although the normal form of PrP (PrP(C)) is monomeric and soluble, it can misfold into a pathogenic form (PrP(Sc)) that has a high content of β-structure and can aggregate forming amyloid fibrils. The mechanism of conversion of PrP(C) into PrP(Sc) is not known but different triggers have been proposed. It can be catalyzed by a PrP(Sc) sample, or it can be induced by an external factor, such as low pH. The pH effect on the structure of PrP was recently studied by computational methods [Campos et al. J. Phys. Chem. B 2010, 114, 12692-12700], and an evident trend of loss of helical structure was observed with pH decrease, together with a gain of β-structures. In particular, one simulation at pH 2 showed an evident misfolding transition. The main goal of the present work was to study the effects of a change in pH to 7 in several transient conformations of this simulation, in order to draw some conclusions about the reversibility of PrP misfolding. Although the most significant effect caused by the change of pH to 7 was a global stabilization of the protein structure, we could also observe that some conformational transitions induced by pH 2 were reversible in many of our simulations, namely those started from the early moments of the misfolding transition. This observation is in good agreement with experiments showing that, even at pH as low as 1.7, it is possible to revert the misfolding process [Bjorndahl et al. Biochemistry 2011, 50, 1162-1173].

A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis.

Conformational Study of GSH and GSSG Using Constant-pH Molecular Dynamics Simulations

Glutathione is a small peptide with a crucial role in living organisms. This molecule is found in Nature in both reduced (GSH) and oxidized (GSSG) forms and a high GSH/GSSG ratio is essential to the cell. Glutathione is also present in several enzymatic reactions and can be found in many protein structures. As small peptides, these molecules do not have a defined structure in solution and are able to sample a broad conformational space. In addition, both molecules have several titration sites (four in GSH and six in GSSG) and their conformational space is inevitably influenced by pH. Here, we present a detailed conformational study of GSH and GSSG in a range of pH values, together with a full pH titration of these molecules. We performed constant-pH MD simulations of GSH and GSSG at 24 pH values in a total of 14.4 μs (300 ns per pH value). We obtained the two titration curves and the pKa values for all titrable groups with good agreement with experimental data. We also observed that GSH and GSSG have a large conformational variability in solution and their structural preferences are not significantly affected upon binding to proteins. Some exceptions were found and investigated in detail.

Coordination-driven self-assembly of thiocyanate complexes of Co(II), Ni(II) and Cu(II) with picolinamide: a structural and DFT study

The new heteroleptic complexes, [M(NCS)2(pia)2] M = Co(II) (1), Ni(II) (2), and [Cu(SCN)2(pia)2] (3), pia = pyridine-2-carboxamide, were synthesized and characterized. Their single crystal X-ray diffraction structures showed octahedral units, with the two thiocyanate ligands occupying cis-positions and binding through the nitrogen atom in Co(II) and Ni(II). In the Cu(II) complex, they were trans and S-bound. The crystal structures display a 2-d structure with NH⋯S hydrogen bonds for the Co(II) and Ni(II), forming tetrameric units, and are isomorphous with the Zn(II) complex, [Zn(NCS)2(pia)2], and a 2-d structure with NH⋯N for the Cu(II). DFT calculations were performed on the new complexes and the analogous polymorphs of [Zn(NCS)2(pia)2], including a second one containing dimeric motifs. The calculated vibrational modes of the thiocyanate ligands corroborate the experimental ones and reflect the coordination mode of the ligand. A comparison between the two Zn(II) polymorphs showed that the NH⋯S bond in the tetramer is stronger (−7.50 kcal mol−1 per metal) than the NH⋯O bond in the dimer (−4.01 kcal mol−1 per metal), indicating a preference for the formation of tetrameric units. The NH⋯N hydrogen bonds calculated in the Cu(II) crystal are stronger (−9.15 kcal mol−1 per metal) than the NH⋯S ones in Ni(II) and Zn(II).

Protonation of DMPC in a Bilayer Environment Using a Linear Response Approximation

pH is a very important property, influencing all important biomolecules such as proteins, nucleic acids, and lipids. The effect of pH on proteins has been the subject of many computational works in recent years. However, the same has not been done for lipids, especially in their most biologically relevant environment: the bilayer. A reason for this is the inherent technical difficulty in dealing with this type of periodic systems. Here, we tackle this problem by developing a Poisson-Boltzmann-based method that takes in consideration the periodic boundary conditions of lipid bilayer patches. We used this approach with a linear response approximation to calculate the pKa value of a DMPC molecule when diluted in zwitterionic lipids. Our results show that DMPC protonation only becomes relevant at quite low pH values (2-3). However, when it happens, it has a strong impact on lipid conformations, leading to significant heterogeneity in the membrane.

pKa Values of Titrable Amino Acids at the Water/Membrane Interface

Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the pK(a) shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pK(a) values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pK(a) values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations.

Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems. These two problems have been circumvented by using ionized (deprotonated) anionic lipids and enough counterions to preserve the electroneutrality. In this work, we propose a method based on the Poisson-Boltzmann equation to estimate the counterion and co-ion concentration close to a lipid bilayer that avoids the need for neutrality at this microscopic level. The estimated number of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC lipid bilayer at different ionization levels. Our results show that the system neutralization represents an overestimation of the number of counterions. Consequently, the resulting lipid bilayer becomes too ordered and practically insensitive to ionization. On the other hand, our proposed approach is able to correctly model the ionization dependent isothermal phase transition of the bilayer observed experimentally. Furthermore, our approach is not too computationally expensive and can easily be used to model diverse charged biomolecular systems in molecular dynamics simulations.

Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers

Current constant-pH molecular dynamics (CpHMD) simulations provide a proper treatment of pH effects on the structure and dynamics of soluble biomolecules like peptides and proteins. However, addressing such effects on lipid membrane assemblies has remained problematic until now, despite the important role played by lipid ionization at physiological pH in a plethora of biological processes. Modeling (de)protonation events in these systems requires a proper consideration of the physicochemical features of the membrane environment, including a sound treatment of solution ions. Here, we apply our recent CpHMD-L method to the study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely reproducing the correct lipid phases of this system, namely, gel-fluid coexistence at pH 4 and a fluid phase at pH 7. A significant transition is observed for the membrane ionization and mechanical properties at physiological pH, providing a molecular basis for the well-established role of phosphatidic acid (PA) as a key player in the regulation of many cellular events. Also, as reported experimentally, we observed pH-induced PA-PA lipid aggregation at acidic pH. By including the titration of anionic phospholipids, the current methodology makes possible to simulate lipid bilayers with increased realism. To the best of our knowledge, this is the first simulation study dealing with a continuous phospholipid bilayer with pH titration of all constituent lipids.

Boron complexes of aromatic ring fused iminopyrrolyl ligands: synthesis, structure, and luminescence properties

The condensation reactions of 2-formylindole (1) or 2-formylphenanthro[9,10-c]pyrrole (2) with various aromatic amines afforded the corresponding phenyl or phenanthrene ring fused mono-/bis-iminopyrrole ligand precursors 3-8, which, upon reaction with BPh3 in an appropriate molar ratio, led to the new mono- and diboron chelate compounds Ph2B[NC8H5C(H)[double bond, length as m-dash]N-2,6-Ar] (Ar = 2,6-iPr2C6H39; C6H510), Ph2B[(NC8H5C(H)[double bond, length as m-dash]N)2-1,4-C6H4]BPh211, Ph2B(NC16H9C(H)[double bond, length as m-dash]N-Ar) (Ar = 2,6-iPr2C6H312; C6H513), and Ph2B[(NC16H9C(H)[double bond, length as m-dash]N)2-1,4-C6H4]BPh214, respectively. Boron complexes 12-14, containing a phenanthrene fragment fused to the pyrrolyl C3-C4 bond, are highly fluorescent in solution, with quantum efficiencies of 37%, 61% and 58% (in THF), respectively, their emission colours ranging from blue to orange depending on the extension of π-conjugation. Complexes 9-11, containing a benzene fragment fused to the pyrrolyl C4-C5 bond, are much weaker emitters, exhibiting quantum efficiencies of 10%, 7% and 6%, respectively. DFT and TDDFT calculations showed that 2,6-iPr2C6H3N-substituents or, to a smaller extent, the indolyl group prevent a planar geometry of the ligand in the excited state and reveal the existence of a low energy weak band in all the indolyl complexes, which is responsible for the different optical properties. Non-doped single-layer light-emitting diodes (OLEDs) were fabricated with complexes 9-14, deposited by spin coating, that of complex 13 revealing a maximum luminance of 198 cd m-2.

Targeting Type 2 Diabetes with C-Glucosyl Dihydrochalcones as Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: Synthesis and Biological Evaluation

Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC50 = 9-23 nM) were considerably weaker inhibitors of SGLT1 (IC50 = 10-19 μM). They showed no effect on the sodium independent GLUT family of glucose transporters, and the most potent ones were not acutely toxic to cultured cells. The interaction of a C-glucosyl dihydrochalcone with a POPC membrane was modeled computationally, providing evidence that it is not a pan-assay interference compound. These results point toward the discovery of structures that are potent and highly selective inhibitors of SGLT2.