Silvia Frutos

Chemical Synthesis of 19F-labeled HIV-1 Protease using Fmoc-Chemistry and ChemMatrix Resin

by Judit Tulla-puche and Silvia Frutos

International Journal of Peptide Research and Therapeutics, 2007

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Genetic Diversity and Gene Flow in Nine-Banded Armadillos in Paraguay

Journal of Mammalogy, 2002

... research. I am grateful to those who assisted me in the field, especially Ismael Mora and Jav... more

Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level

by Silvia Frutos and Blanca Rey-Serra

Biophysical Journal, 2016

The unfolding and folding of protein barnase has been extensively investigated in bulk conditions... more The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.

Cooperativas de trabajo y pequeñas empresas solidarias en la micro-región de Paraná (Argentina)

Ciencia Docencia Y Tecnologia, May 1, 2008

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Identification by19F NMR of Traditional Chinese Medicinal Plants Possessing Prolyl Oligopeptidase Inhibitory Activity

Chembiochem, 2006

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides... more Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy termini of the proline residues. This peptidase has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and might therefore have important clinical implications. Traditional Chinese medicinal (TCM) plants provide a rich source of unexplored compounds for strategies to find novel POP inhibitors, but the traditional methodologies used to identify POP inhibitors could have some limitations when working with natural products: interference with the colorimetric or fluorimetric detection methods commonly used to screen for POP inhibitors can result in the generation of false positives or false negatives. Since NMR screening is less prone to such interference, we decided to explore the use of 19F NMR to screen for POP inhibitors. We synthesized a new 19F-labeled POP substrate--Z-Gly-Pro-Phe-4(CF3)-NH2--and used it to search for new POP inhibitors in TCM plant extracts. We identified several plants with high POP-inhibitory activity and show here that the combination of 19F NMR and TCM plant extracts is a useful tool for identifying new POP inhibitors.

A Fast and Robust19F NMR-Based Method for Finding New HIV-1 Protease Inhibitors

Cheminform, 2006

The human immunodeficiency virus (HIV) which encodes, among other indispensable enzymes, an aspar... more The human immunodeficiency virus (HIV) which encodes, among other indispensable enzymes, an aspartic protease that is essential for viral maturation and replication. Numerous inhibitors of the protease have been developed. However, the eventual resistance of HIV-1 to these drugs implies a continuous battle to develop new inhibitors. Proposed herein is a robust, fast, and reliable method employing (19)F NMR for the evaluation of the inhibitory activity of new compounds against HIV-1 protease.

Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

by Judit Tulla-puche and Silvia Frutos

Nucleic Acids Research, 2015

DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA ima... more DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bisintercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel singlemolecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.

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Structure of the branched intermediate in protein splicing

by Silvia Frutos and M. Vila-Perello

Proceedings of the National Academy of Sciences, 2014

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner... more Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use proteinengineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogenbond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site. expressed | protein semisynthesis P rotein splicing is a posttranslational modification in which an internal domain, termed an intein, excises itself from a host protein with concomitant ligation of the flanking sequences (termed the N-and C-exteins) (1, 2). Inteins are found in unicellular organisms from all domains of life (3) and belong to the HINT (Hedgehog INTein) superfamily of autoprocessing domains (4), which includes the cholesterol ligase domain found in the eponymous hedgehog family of developmental proteins present in all bilaterian animals (5, 6). High-resolution structures of inteins reveal a characteristic horseshoe-like β-sheet fold , common to all HINT family members (8), which positions catalytic residues from four conserved sequence blocks (A, B, F, G) in proximity to N-and C-terminal splice junctions . This structural information has aided mechanistic studies into the protein splicing process, which we know to be a multistep cascade involving a series of acyl-transfer reactions . Although a biological role for protein splicing remains elusive, an ever-deepening understanding of the splicing mechanism has led to the development of a wide range of biotechnology and chemical biology approaches based on engineered inteins (9).

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Applications of 3-aminolactams: design, synthesis, and biological evaluation of a library of potential dimerisation inhibitors of HIV1-protease

Organic & Biomolecular Chemistry, 2012

In the context of our studies on the applications of 3-aminolactams as conformationally restricte... more In the context of our studies on the applications of 3-aminolactams as conformationally restricted pseudodipeptides, we report here the synthesis of a library of potential dimerisation inhibitors of HIV1-protease. Two of the pseudopeptides were active on the wild type virus (HIV1) at micromolar levels (EC(50)). Although the peptides showed lower anti-viral activity than previously reported dimerisation inhibitors, our results demonstrate that the piperidone moiety does not prevent cell penetration, and hence that such derivatization is compatible with potential anti-HIV treatment.

Branched intermediate formation stimulates peptide bond cleavage in protein splicing

Nature Chemical Biology, 2010

Protein splicing is a posttranslational modification in which an intein domain excises itself out... more Protein splicing is a posttranslational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mxe GyrA intein using protein semi-synthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond cleaved during succinimide formation. We propose that this structural change reflects a re-organization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction.

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Identification by 19F NMR of Traditional Chinese Medicinal Plants Possessing Prolyl Oligopeptidase Inhibitory Activity

ChemBioChem, 2006

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides... more Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy termini of the proline residues. This peptidase has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and might therefore have important clinical implications. Traditional Chinese medicinal (TCM) plants provide a rich source of unexplored compounds for strategies to find novel POP inhibitors, but the traditional methodologies used to identify POP inhibitors could have some limitations when working with natural products: interference with the colorimetric or fluorimetric detection methods commonly used to screen for POP inhibitors can result in the generation of false positives or false negatives. Since NMR screening is less prone to such interference, we decided to explore the use of 19F NMR to screen for POP inhibitors. We synthesized a new 19F-labeled POP substrate--Z-Gly-Pro-Phe-4(CF3)-NH2--and used it to search for new POP inhibitors in TCM plant extracts. We identified several plants with high POP-inhibitory activity and show here that the combination of 19F NMR and TCM plant extracts is a useful tool for identifying new POP inhibitors.

Cover Picture: Identification by 19F NMR of Traditional Chinese Medicinal Plants Possessing Prolyl Oligopeptidase Inhibitory Activity (ChemBioChem 5/2006)

ChemBioChem, 2006

Disruption of the HIV-1 protease dimer with interface peptides: Structural studies using NMR spectroscopy combined with [2-13C]-Trp selective labeling

Biopolymers, 2007

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of ga... more HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a fourstranded antiparallel b-sheet involving the N-and Ctermini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer.

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A fast and robust 19F NMR-based method for finding new HIV-1 protease inhibitors

Bioorganic & Medicinal Chemistry Letters, 2006

The human immunodeficiency virus (HIV) which encodes, among other indispensable enzymes, an aspar... more The human immunodeficiency virus (HIV) which encodes, among other indispensable enzymes, an aspartic protease that is essential for viral maturation and replication. Numerous inhibitors of the protease have been developed. However, the eventual resistance of HIV-1 to these drugs implies a continuous battle to develop new inhibitors. Proposed herein is a robust, fast, and reliable method employing (19)F NMR for the evaluation of the inhibitory activity of new compounds against HIV-1 protease.

Peptidomimetic Dimerization Inhibitors of HIV-1 Protease: Further Insights into Structural Variations and Mechanism of Action

Antiviral Research, 2007

Background: Although clinically effective when used in combination, current HIV-1 therapies are l... more

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Electrostatic Binding and Hydrophobic Collapse of Peptide–Nucleic Acid Aggregates Quantified Using Force Spectroscopy

ACS Nano, 2013

Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or... more

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