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Annalisa Pastore

Publications and source records attributed to Annalisa Pastore.

At least 37 records · Page 2Linked to original sources
PubMed2004

The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability.

Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding. Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans. Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps. Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities. Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange. By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins. Thermal stability is also gained by the addition of Fe(2+). This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations. We discuss the implications that our findings have for the role of frataxins as iron-binding proteins.

Amino Acid Sequence↗
PubMed2004

Ab initio study of NMR 15N chemical shift differences induced by Ca2+ binding to EF-hand proteins.

EF-hands are Ca(2+) binding motifs that are widely distributed throughout the entire living organism kingdom. At present, relatively little is known at a quantum mechanical level about the mechanisms that allow Ca(2+) to be recognized specifically by EF-hands and to induce a conformational switch from a compact ("closed") conformation to an "open" state that exposes a large patch of hydrophobic residues. Here, we present a study of NMR (15)N chemical shifts based on ab initio quantum mechanical calculations carried out on a minimalist model system linking both Ca(2+) binding sites across the beta-sheet of an EF-hand domain. Calculated and experimentally determined chemical shift changes are correlated with structural changes induced upon metal binding. The effect of Ca(2+) binding on these (15)N shifts can be dissected into two main contributions: one from pi-polarization of beta-sheet amide groups and the other from rotation of an isoleucine side chain. By correlating this description with experimental evidence, different polarization states for the beta-sheet amide groups were identified and linked to the overall conformation of different EF-hand domains. When all four beta-sheet amide groups are polarized, the ab initio calculations in our model indicate a cooperative stabilization effect due to the establishment of a circular network of donor-acceptor interactions connecting the two Ca(2+) ions across the beta-sheet. The emerging hypothesis from our analysis is that this cooperative network of interactions is essential for stabilizing the "open" conformation of an EF-hand domain.

Amino Acid Motifs↗
PubMed2004

Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement.

Confinement of a protein in a small inert space and microviscosity are known to increase its thermodynamic stability in a way similar to the mechanisms that stabilize protein fold in the cell. Here, to examine the influence of confinement on protein stability we choose four test cases of single domain proteins characterized by a wide range of melting temperatures, from approximately 73 degrees C of titin I27 to approximately 36 degrees C of yeast frataxin. All proteins are stabilized when confined in the gel, the most dramatic stabilization being that of yeast frataxin, whose melting temperature increased by almost 5 degrees C in the gel. In addition to being simple to use, this approach allows us to change the viscosity of the solvent without changing its composition or altering the structure of the proteins. The dimensions of the pores of the gels fall in the nanometer range, hence they are similar to those of the chaperone cavity. This method could therefore be used as a novel and powerful approach for protein folding studies.

Acrylic Resins↗
PubMed2004

Solution structure of ZASP PDZ domain; implications for sarcomere ultrastructure and enigma family redundancy.

Z band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of alpha-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined the structure of ZASP PDZ by NMR and showed that it is a classical class 1 PDZ domain that recognizes the carboxy-terminal sequence of an alpha-actinin-2 calmodulin-like domain with micromolar affinity. We also characterized the role of each component in the ternary complex ZASP/alpha-actinin-2/titin, showing that the alpha-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that recognized by the titin Z repeats. ZASP PDZ structure was used to model other members of the enigma family by homology and to predict their abilities to bind alpha-actinin-2.

Actinin↗
PubMed2004

Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites.

CyaY is the bacterial ortholog of frataxin, a small mitochondrial iron binding protein thought to be involved in iron sulphur cluster formation. Loss of frataxin function leads to the neurodegenerative disorder Friedreich's ataxia. We have solved the solution structure of CyaY and used the structural information to map iron binding onto the protein surface. Comparison of the behavior of wild-type CyaY with that of a mutant indicates that specific binding with a defined stoichiometry does not require aggregation and that the main binding site, which hosts both Fe(2+) and Fe(3+), occupies a highly anionic surface of the molecule. This function is conserved across species since the corresponding region of human frataxin is also able to bind iron, albeit with weaker affinity. The presence of secondary binding sites on CyaY, but not on frataxin, hints at a possible polymerization mechanism. We suggest mutations that may provide further insights into the frataxin function.

Amino Acid Sequence↗
PubMed2004

NMR and molecular dynamics studies of the interaction of melatonin with calmodulin.

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca(2+) concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring (15)N and (1)H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca(2+) by paramagnetic Tb(3+) allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.

Binding Sites↗
PubMed2004

Bacterial IscU is a well folded and functional single domain protein.

Iron-sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an alphabeta fold with high alpha-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the (1)H-(15)N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron-sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction.

Escherichia coli Proteins↗
PubMed2004

Interaction of sweet proteins with their receptor. A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin.

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of "sweet fingers", i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential "sweet fingers" of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37-47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the "wedge model", recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett.526, 1-3.].

Binding Sites↗
PubMed2004

The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module.

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using (15)N NMR relaxation collected at two (15)N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.

Algorithms↗
PubMed2003

Domain architecture of the polyglutamine protein ataxin-3: a globular domain followed by a flexible tail.

Anomalous expansion of a polyglutamine (polyQ) tract in the protein ataxin-3 causes spinocerebellar ataxia type 3, an autosomal dominant neurodegenerative disease. Very little is known about the structure and the function of ataxin-3, although this information would undoubtedly help to understand why the expanded protein forms insoluble nuclear aggregates and causes neuronal cell death. With the aim of establishing the domain architecture of ataxin-3 and the role of the polyQ tract within the protein context, we have studied the human and murine orthologues using a combination of techniques, which range from limited proteolysis to circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The two protein sequences share a highly conserved N-terminus and differ only in the length of the glutamine repeats and in the C-terminus. Our data conclusively indicate that ataxin-3 is composed by a structured N-terminal domain, followed by a flexible tail. Moreover, [(15)N]glutamine selectively labelled samples allowed us to have a direct insight by NMR into the structure of the polyQ region.

Amino Acid Sequence↗
PubMed2003

82-FIP, a novel FMRP (fragile X mental retardation protein) interacting protein, shows a cell cycle-dependent intracellular localization.

FMRP is an RNA binding protein whose absence produces pathological manifestations of the fragile-X syndrome. FMRP is a component of mRNP complexes found in association with actively translating polyribosomes, RNA complexes trafficking in neurites, RNA granules in cytoplasm and, in Drosophila, with the RNAi machinery. We report here the identification and characterization of a novel FMRP-interacting protein associated to polyribosomes as a component of mRNP complexes containing FMRP. We named this protein 82-FIP (82-kD FMRP Interacting Protein). FMRP interacts with 82-FIP through a novel interaction motif located in its N-terminal region. The distribution of 82-FIP in different areas of the brain is very similar to that of FMRP. However, unlike FMRP, 82-FIP is found in both nucleus and cytoplasm in some neurons, while it appears only cytoplasmic in others. Subcellular distribution of 82-FIP is cell cycle-dependent in cultured cells, suggesting that the composition of some FMRP-containing RNP complexes may be cell cycle-modulated.

Carrier Proteins↗
PubMed2003

From Alzheimer to Huntington: why is a structural understanding so difficult?

An increasing family of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, prion encephalopathies and cystic fibrosis is associated with aggregation of misfolded polypeptide chains which are toxic to the cell. Knowledge of the three-dimensional structure of the proteins implicated is essential for understanding why and how endogenous proteins may adopt a non-native fold. Yet, structural work has been hampered by the difficulty of handling proteins insoluble or prone to aggregation, and at the same time that is why it is interesting to study these molecules. In this review, we compare the structural knowledge accumulated for two paradigmatic misfolding disorders, Alzheimer's disease (AD) and the family of poly-glutamine diseases (poly-Q) and discuss some of the hypotheses suggested for explaining aggregate formation. While a common mechanism between these pathologies remains to be proven, a direct comparison may help in designing new strategies for approaching their study.

Alzheimer Disease↗
PubMed2003

The role of a clinically important mutation in the fold and RNA-binding properties of KH motifs.

We have investigated the role in the fold and RNA-binding properties of the KH modules of a hydrophobic to asparagine mutation of clinical importance in the fragile X syndrome. The mutation involves a well-conserved hydrophobic residue close to the N terminus of the second helix of the KH fold (alpha2(3) position). The effect of the mutation has been long debated: Although the mutant has been shown to disrupt the three-dimensional fold of several KH domains, the residue seems also to be directly involved in RNA binding, the main function of the KH module. Here we have used the KH3 of Nova-1, whose structure is known both in isolation and in an RNA complex, to study in detail the role of the alpha2(3) position. A detailed comparison of Nova KH3 structure with its RNA/KH complex and with other KH structures suggests a dual role for the alpha2(3) residue, which is involved both in stabilizing the hydrophobic core and in RNA contacts. We further show by nuclear magnetic resonance (NMR) studies in solution that L447 of Nova-1 in position alpha2(3) is in exchange in the absence of RNA, and becomes locked in a more rigid conformation only upon formation of an RNA complex. This implies that position alpha2(3) functions as a "gate" in the mechanism of RNA recognition of KH motifs based on the rigidification of the fold upon RNA binding.

Amino Acid Motifs↗