Margarida Gairí

Molecular recycling within amyloid fibrils

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of β-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation

Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimers disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.

Structural characterization of the natively unfolded N-terminal domain of human c-Src kinase: insights into the role of phosphorylation of the unique domain.

The N-terminal regions of the members of Src family of non-receptor protein tyrosine kinases are intrinsically unfolded and contain the maximum sequence divergence among them. In this study, we have addressed the structural characterization by nuclear magnetic resonance of this region of 84 residues that encompasses the SH4 and the unique domains (USrc) of the human c-Src. With this aim, the backbone assignment was performed using (13)C-detected experiments that overcome the spectral resolution problems and the large number of prolines that are typical for intrinsically unfolded proteins. The analysis of the residual dipolar couplings measured for the USrc indicates the presence of a low populated helical structure in the 60-75 region. No long-range contacts between remote fragments of the chain were detected with paramagnetic relaxation enhancement experiments. The structural characterization was extended to two different phosphorylation states of USrc that encompassed three different phosphorylated sites, Ser17, Thr37, and Ser75. The structural and conformational changes upon phosphorylation were monitored through chemical shift perturbations and residual dipolar couplings, indicating that modifications occur at local level and no global rearrangements were apparent. These results suggest a scenario where phosphorylation induces a global electrostatic perturbation that could be involved in the membrane unbinding of c-Src and that could be related with the localization of the enzyme. These observations suggest the unique domain of Src kinases as a source of selectivity and reinforce the relevant role of intrinsically disordered proteins in biological processes.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism.

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

Use of BOP reagent for the suppression of diketopiperazine formation in Boc/Bzl solid-phase peptide synthesis

Abstract BOP coupling reagent suppresses the formation of diketopiperazines in the solid-phase coupling of the third amino acid to dipeptides when a nitro-benzylic “handle” is used to link the peptide to the resin. The amino function of the second amino acid may be deprotected with TFA and the coupling carried out without a prior neutralisation step.

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

Significance Numerous reports indicate that amyloid-β peptide (Aβ) oligomers, considered the pathogenic molecular form of Aβ in Alzheimer´s disease (AD), exert their neurotoxicity within the membrane. Therefore, it is critical to characterize them in such an environment. Here, we worked with two major Aβ variants and handled them as if they were membrane proteins. By doing so, we found that the Aβ variant most strongly linked to AD assembled into stable Aβ oligomers that adopted a specific structure and incorporated into membranes as pores, a feature linked to neurotoxicity. Having access to pore-forming Aβ oligomers with such a specific structure offers unique opportunities to fully characterize them and establish their involvement in AD. The formation of amyloid-β peptide (Aβ) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer’s disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aβ variants, Aβ40 and Aβ42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aβ in lipid environments—namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aβ40 and Aβ42 showed different behavior. Aβ40 aggregated into amyloid fibrils, whereas Aβ42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a β-barrel arrangement that we named β-barrel pore-forming Aβ42 oligomers (βPFOsAβ42). Because Aβ42, relative to Aβ40, has a more prominent role in AD, the higher propensity of Aβ42 to form βPFOs constitutes an indication of their relevance in AD. Moreover, because βPFOsAβ42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of βPFOs and, more generally, membrane-associated Aβ oligomers in AD.

3D structure of kaliotoxin: is residue 34 a key for channel selectivity?

Kaliotoxin (KTX) is a natural peptide blocker of voltage‐dependent K+ channels. The 3D structure of a truncated analogue of KTX (Fernández et al. (1994) Biochemistry 33, 14256–14263) was determined by NMR spectroscopy and showed significant differences from structures established for other related scorpion toxins. A recent publication with the structure of the complete toxin (Aiyar et al. (1995) Neuron 15, 1169–1181) did not confirm these differences. In this communication we report NMR data for KTX at pH 3.0, 5.5 and 7.2 and the 3D structure obtained from data at pH=5.5. Complete KTX displays a folding similar to that of other toxins with an α‐helix and a β‐sheet linked by two disulphide bonds. The pKa of His 34 is anomalously low (4.7–5.2 depending on the buffer) owing to its interaction with two Lys residues (including the essential Lys 27), the charged N‐terminus and the side chain of Met 29. Charged residues are placed symmetrically with respect to an axis that approximately coincides with one of the principal components of the moment of inertia of the toxin. His 34, which occupies a well‐defined position between two conserved Cys, is located on the centre of a layer of charged groups. Positively and negatively charged residues are found at the same position in related toxins. It is suggested that electrostatic effects modulate the distances between positive charges in flexible side chains, contributing to the fine tuning of the selectivity toward different channel subclasses and that the approximate coincidence between the moment of inertia and the charge axis facilitate the approach of the toxin to the channel. The very low pKa of His 34 implies that it will be completely unprotonated at physiological pH.

Convergent solid-phase peptide synthesis. X. Synthesis and purification of protected peptide fragments using the photolabile Nbb-resin☆☆☆

Abstract Protected peptide fragments corresponding to the 12–18, 31–38 and 59–67 segments of the Uteroglobin monomer have been synthesised on a solid support using the photolabile ortho -nitrobenzyl unit as a handle. Attachment of the first amino acid of the sequence has been carried out in three different ways and a new procedure for avoiding the formation of DKPs in the coupling of the third amino acid with this resin-handle is described. Photolytic detachment of the peptides from the solid support occurs in good yields. In spite of their low solubility in the normal solvents used in reverse-phase MPLC and HPLC techniques, protected peptide fragments can be purified by MPLC utilising solvents containing a high proportion of DMF.

A new side opening on prolyl oligopeptidase revealed by electron microscopy

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.

Convergent solid-phase peptide synthesis. XI. Synthesis and purification of protected peptide segments spanning the entire sequence of the uteroglobin monomer using the photolabile nbb-resin.

Abstract Protected peptide segments corresponding to the 1–4, 5–11, 19–30, 39–49 and 50–58 segments of the Uteroglobin monomer have been synthesised on a solid support using the photolabile ortho -nitrobenzyl unit as a handle. Photolytic detachment of the peptides from the solid support occurred in good yields. The 50–58 protected peptide segment has also been synthesised using the base-labile NPE handle, and detached from the solid support in excellent yield by treatment with a 20% solution of piperidine in DMF. The protected peptide segments were purified by MPLC and/or preparative HPLC, using solvents containing DMF.