Luisa Ronga

Does tetracycline bind helix 2 of prion? An integrated spectroscopical and computational study of the interaction between the antibiotic and α helix 2 human prion protein fragments

We demonstrate here that tetracycline (TC) can strongly interact (KD′ = 189 ± 7 nM) with model peptides derived from the C‐terminal globular domain of the prion protein, hPrP [173‐195], and that interaction concerns residues within the C‐terminal half of the helix 2, a short region previously indicated as endowed with ambivalent conformational behavior and implicated in PrP conversion to the β‐sheet‐rich, infective scrapie variant. Data have been confirmed by binding studies with the N‐terminal truncated 180‐195 variant that displays a dissociation constant of 483 ± 30 nM. Remarkably, TC does not influence the structure of the N‐terminally fluoresceinated peptides that both show α‐helical conformations. Docking calculations and molecular dynamics simulations suggest a direct, strong interaction of the antibiotic with exposed side chain functional groups of threonines 190‐193 on the solvent‐exposed surface of helix 2. Proteins 2007

The prion protein: structural features and related toxic peptides

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrPC) into an insoluble, partially protease‐resistant abnormal scrapie form (PrPSc). PrPC is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrPC to PrPSc parallels a conformational change of the polypeptide from a predominantly α‐helical to a highly β‐sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure‐activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N‐terminal and globular C‐terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high β‐sheet propensity and tendency to form β‐sheet‐like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.

Conformational Diseases and Structure-Toxicity Relationships: Lessons from Prion-Derived Peptides

The physiological form of the prion protein is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. Available evidence suggests that this protein is essential for neuronal integrity in the brain, possibly with a role in copper metabolism and cellular response to oxidative stress. In prion diseases, the benign cellular form of the protein is converted into an insoluble, protease-resistant abnormal scrapie form. This conversion parallels a conformational change of the polypeptide from a predominantly alpha-helical to a highly beta-sheet secondary structure. The scrapie form accumulates in the central nervous system of affected individuals, and its protease-resistant core aggregates into amyloid fibrils outside the cell. The pathogenesis and molecular basis of the nerve cell loss that accompanies this process are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. A large amount of structure-activity studies is based on the prion fragment approach, but the resulting information is often difficult to untangle. This overview focuses on the most relevant structural and functional aspects of the prion-induced conformational disease linked to peptides derived from the unstructured N-terminal and globular C-terminal domains.

NMR structure and CD titration with metal cations of human prion alpha2-helix-related peptides.

The 173–195 segment corresponding to the helix 2 of the C-globular prion protein domain could be one of several “spots” of intrinsic conformational flexibility. In fact, it possesses chameleon conformational behaviour and gathers several disease-associated point mutations. We have performed spectroscopic studies on the wild-type fragment 173–195 and on its D178N mutant dissolved in trifluoroethanol to mimic the in vivo system, both in the presence and in the absence of metal cations. NMR data showed that the structure of the D178N mutant is characterized by two short helices separated by a kink, whereas the wild-type peptide is fully helical. Both peptides retained these structural organizations, as monitored by CD, in the presence of metal cations. NMR spectra were however not in favour of the formation of definite ion-peptide complexes. This agrees with previous evidence that other regions of the prion protein are likely the natural target of metal cation binding.

Structural characterization of a neurotoxic threonine-rich peptide corresponding to the human prion protein α2-helical 180–195 segment, and comparison with full-length α2-helix-derived peptides†

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrPC full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrPC makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright

Supported oligomethionine sulfoxide and Ellman’s reagent for cysteine bridges formation

A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide. Supported reagents advantageously simplify the work-up of such disulfide bond formation, but may lead to a significant decrease in yield of the oxidized product. In this study, two resins working through different mechanisms were evaluated: Clear-Ox, a supported version of Ellman’s reagent and Oxyfold, consisting in a series of oxidized methionine residues. The choice of the supported reagent is discussed on the light of reaction speed, side-products formation and yield considerations.

Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6).

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107‐amino‐acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X‐ray diffraction. The refolded enzyme was compared to the recombinant, wild‐type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N‐terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure‐function relationship of enzymes reachable by complete chemical synthesis.

Peptide Fragment Approach to Prion Misfolding: The Alpha-2 Domain

The mechanism that underlies a multitude of human disorders, including type II diabetes, Parkinson’s, Huntington’s and Alzheimer’s, and the prion encephalopathies, is β-structure expansion through a pathogenic aggregation-prone monomeric form. β-sheet expansion disorders share intermolecular association as a common determinant, being therefore collectively identified as conformational diseases, but little is known about the underlying mechanism. Transmissible spongiform encephalopathies, also known as prion diseases, are all characterised by progressive neuronal degeneration associated to marked extracellular accumulation of an amyloidogenic conformer of the normal cellular prion protein (PrPC), referred to as the scrapie isoform (PrPSc), which is thought to be responsible for the disease symptoms. PrPC is a ubiquitous 231-amino acid glycoprotein, whose physiological role is still elusive. It is organised as an N-terminal disordered region and a compact C-terminal domain, where secondary structure elements consist of three α-helices (α1, α2 and α3), with an α2-α3 disulphide bridge, and two short β-strands (β1 and β2). Evidence accumulated so far suggests that the protein possesses one or several ‘spots’ of intrinsic conformational weakness, which may trigger generic folding, leading the whole architecture to adopt aggregation-prone conformations. One of such spots is suspected to be the C-terminal side of the α-helix 2, which has recently gained the attention of several investigations because it gathers several disease-associated point mutations, can be strongly fibrillogenic and toxic to neuronal cells, and possesses chameleon conformational behaviour. This paper briefly reviews recent literature on α-2 domain-derived model peptides.