Rui M. M. Brito

Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine

The fluorescence quantum yield of N-phenyl-1-naphthylamine (NPN) increases about 10-fold and the wavelength of maximum fluorescence emission is blue-shifted when this molecule partitions into the apolar core of micellar structures from the aqueous phase. This property allowed the utilization of NPN as a fluorescent indicator of micelle formation by 14 different surfactants belonging to the families of alkyltrimethylammonium halides, alkylsulfates, alkylbetaines, alkylglucosides, and bile salts. The critical micelle concentrations (CMCs) determined with NPN agreed well with literature values. In this work NPN was used at a concentration of 10(-6) M which allowed determination of CMCs in the range between approximately 10(-5) and greater than 10(-2) M. With high-sensitivity instrumentation considerably lower NPN concentrations can be used and consequently considerably lower CMCs can be rapidly and accurately determined.

The Tetrameric Protein Transthyretin Dissociates to a Non-native Monomer in Solution A NOVEL MODEL FOR AMYLOIDOGENESIS

In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimers disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett.418, 297–300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions.

The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution

Amyloid fibril formation and deposition are the basis for a wide range of diseases, including spongiform encephalopathies, Alzheimers and familial amyloidotic polyneuropathies. However, the molecular mechanisms of amyloid formation are still poorly characterised. In certain forms of familial amyloidotic polyneuropathy (FAP), the amyloid fibrils are mostly constituted by variants of transthyretin (TTR). V30M‐TTR is the most frequent variant, and L55P‐TTR is the variant associated with the most aggressive form of amyloidosis. Here, we report gel filtration chromatography experiments to characterise the aggregation states of WT‐, V30M‐, L55P‐TTR and a non‐amyloidogenic variant, T119M‐TTR, in solution, at nearly physiological pH. These studies show that all four protein tetramers dissociate to monomer upon dilution, in the sub‐micromolar range, at pH 7.0. The amyloidogenic proteins V30M‐ and L55P‐TTR show a complex equilibrium between monomers, tetramers and high molecular weight aggregate species. These aggregates dissociate directly to monomer upon dilution. This study shows that the tendency to form aggregates among the four studied proteins correlates with their known amyloidogenic potential. Thus, the amyloidogenic mutations could perturb the structure and/or stability of the monomeric species leading initially to the formation of soluble aggregates and at a later stage to insoluble amyloid fibrils.

Mining approximate motifs in time series

The problem of discovering previously unknown frequent patterns in time series, also called motifs, has been recently introduced. A motif is a subseries pattern that appears a significant number of times. Results demonstrate that motifs may provide valuable insights about the data and have a wide range of applications in data mining tasks. The main motivation for this study was the need to mine time series data from protein folding/unfolding simulations. We propose an algorithm that extracts approximate motifs, i.e. motifs that capture portions of time series with a similar and eventually symmetric behavior. Preliminary results on the analysis of protein unfolding data support this proposal as a valuable tool. Additional experiments demonstrate that the application of utility of our algorithm is not limited to this particular problem. Rather it can be an interesting tool to be applied in many real world problems.

Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4.

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin‐4 (IL4), a four‐helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T‐IL4 and C24T‐IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent‐accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild‐type protein. Transition temperatures of unfolding decreased 14°C for C3T‐IL4 and 10°C for C24T‐IL4, when compared to WT‐IL4, and the conformational stability, at 25°C, decreased 4.9 kcal/mol for C3T‐IL4 and 3.2 kcal/mol for C24T‐IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.

Comparative calorimetric study of non-amyloidogenic and amyloidogenic variants of the homotetrameric protein transthyretin.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of amyloidosis involving amino acid substitutions in transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of FAP. The thermal stability of the wild-type, V30M-TTR, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal unfolding of TTR is a spontaneous reversible process involving a highly co-operative transition between folded tetramers and unfolded monomers. All variants of transthyretin are very stable to the thermal unfolding that occurs at very high temperatures, most probably because of their oligomeric structure. The data presented in this work indicated that for the homotetrameric form of the wild-type TTR and its variants, the order of stability is as follows: wild-type TTR approximately > T119M-TTR > L55P-TTR > V30M-TTR, which does not correlate with their known amyloidogenic potential.

“Structural characterization of the minimal segment of TDP-43 competent for aggregation”

TDP-43 is a nuclear protein whose abnormal aggregates are implicated in ALS and FTLD. Recently, an Asn/Gln rich C-terminal segment of TDP-43 has been shown to produce aggregation in vitro and reproduce most of the proteins pathological hallmarks in cells, but little is known about this segments structure. Here, CD and 2D heteronuclear NMR spectroscopies provide evidence that peptides corresponding to the wild type and mutated sequences of this segment adopt chiefly disordered conformations that, in the case of the wild type sequence, spontaneously forms a β-sheet rich oligomer. Moreover, MD simulation provides evidence for a structure consisting of two β-strands and a well-defined, yet non-canonical structural element. Furthermore, MD simulations of four pathological mutations (Q343R, N345K, G348V and N352S) occurring in this segment predict that all of them could affect this regions structure. In particular, the Q343R variant tends to stabilize disordered conformers, N345K permits the formation of longer, more stable β-strands, and G348V tends to shorten and destabilize them. Finally, N352S acts to alter the β-stand register and when S352 is phosphorylated, it induces partial unfolding. Our results provide a better understanding of TDP-43 aggregation process and will be useful to design effectors capable to modulate its progression.

Changes in channel trafficking and protein stability caused by LQT2 mutations in the PAS domain of the HERG channel.

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay.

Biological activity of heterologous murine interleukin-10 and preliminary studies on the use of a dextrin nanogel as a delivery system

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer with two intramolecular disulphide bonds essential for its biological activity. A mutated form of murine IL-10 was successfully expressed in E. coli, recovered and purified from inclusion bodies. Its ability to reduce tumor necrosis factor α synthesis and down-regulate class II major histocompatibility complex molecules expression on endotoxin-stimulated bone marrow-derived macrophages was confirmed, and shown to be similar to that of a commercially available IL-10. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems that can effectively deliver the protein. In this work it is shown that a dextrin nanogel effectively incorporate IL-10, stabilize, and enable the slow release of biologically active IL-10 over time. Altogether, these results demonstrate the suitability of dextrin nanogel to be used as a system for the controlled release of IL-10.

Amyloid Formation by Transthyretin: From Protein Stability to Protein Aggregation

In recent years the issues of protein stability, folding and aggregation have become central in several pathological conditions and in particular in amyloid diseases. Here, we review the recent developments on the molecular mechanisms of amyloid formation by transthyretin (TTR), in particular, in what concerns to protein conformational stability, protein folding and aggregation. Transthyretin has been implicated in pathologies such as senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) which are characterized by extracellular deposition of insoluble amyloid fibrils. SSA is generally a mild disorder and affects predominantly individuals over 80 years of age. In contrast, FAP is an autossomal dominant lethal disease, characterized by peripheral neuropathy, which may affect individuals from their twenties. While in SSA WT-TTR and its fragments are the major constituents of the amyloid fibrils, in FAP and FAC the amyloid fibrils are mostly constituted by variants of TTR. Today, more than 80 amyloidogenic TTR mutations throughout the TTR sequence are known. Transthyretin is a homotetrameric protein found in the plasma and in the cerebral-spinal fluid, it is synthesized in the liver and in the choroid plexus of the brain, it has a total molecular mass of 55kDa and a high percentage of β-sheet. Current views on amyloid fibril formation by TTR state that, depending on the protein variant or solution conditions, the native tetrameric protein might dissociate to non-native or partially unfolded monomeric (or even dimeric) species with a high tendency for ordered aggregation into soluble oligomers which grow into insoluble oligomers and eventually mature amyloid fibrils. Thus, issues such as dissociation thermodynamics and dissociation kinetics of the native tetrameric TTR and thermodynamic stability and conformational fluctuations of the non-native TTR molecular species are essential in determining the amyloidogenic potential of different TTR variants. In addition, several other cellular and tissue factors must be involved in modulating the penetrance and age of onset of amyloid pathologies by TTR.