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Resonance in Biology

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  • Venue: (Seminar room, first floor) Collegio A Volta, Pavia
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Resonance in Biology

When: Pavia 13th-14th September 2019

Where: Volta College, Seminar room, first floor

Download the event poster here: resonance_poster_nero.pdf (93 downloads) .

The event is free but you are kindly requested to register in Eventbrite

Why: We are holding a meeting in Pavia on the 13th and 14th of September to have ways to present applications of Biological nuclear magnetic resonance (NMR), a technique which will soon be represented in Pavia thanks to the arrival of the new 700 MHz Instrument.

Pavia is not new to this technique: Luigi Giulotto (1911-1986), a physics professor of University of Pavia, carried out seminal experiments to detect the NMR signal in liquid samples immediately after the discovery of NMR by Felix Bloch and Edward Mills Purcell in the immediate post-war period. Giulotto created around him a group of valid experimental physicists who obtained important results in nuclear and molecular spectroscopy. Giulotto’s work brought Pavia to a level of international visibility and established Pavia University as the first Italian research center in this sector.

The September meeting is meant to celebrate the 80th birthday of Pierandrea Temussi, the founder, together with Anna Laura Segre, of NMR in Italy. After getting his degree in Naples he entered very early on at the same University first as an assistant and later on as associated and then full professor. Temussi left to the States in 1965-66 where he worked in Urbana-Champain, Illinois, in the group of Prof. Gutowsky, the first to apply NMR methods to the field of chemistry. They published together Gutowsky, H. S. and Temussi, P. A. (1967) Conformational Isomerization of Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine. J. Amer. Chem. Soc. 89, 4358. In 1967, Temussi spent a short period in Israel in Prof. Lifson’s group at the Weizman, just in time to see the end of the six days war. The first publications in NMR came around 1973 (Bradbury, E. M., Crane-Robinson, C., Paolillo, L. and Temussi, P. A. (1973) NMR Studies of a Polypeptide in a Non-protonating Solvent System. J. Amer. Chem. Soc. 95, 1638 and Bradbury, E. M., Crane-Robinson, C., Paolillo, L. and Temussi, P. A. (1973) NMR Studies of the Helix-Coil Transition of Polypeptides in Non-protonating Solvent Mixtures Polymer 14, 303). In 1976, he studied the structure of aspartame, immediately after the dipeptide was introduced as a sweetener. This was only the beginning of more than 30 years studies on biological active peptides. At the end of the ‘90ies, Temussi moved to study proteins, focusing mostly on the sweet protein monellin. Over the last 15 years, his main topic of interest is the study of crowding and confinements by NMR, a field in which he has published more than 20 articles

This event is under the auspices of the Initiative for Development and Engineering of Antibodies (IDEA) and was sponsored by the Frontiers journal and Bruker Italy.

Resonance in Biology Workshop

Day 1 – 13th of September

14.00-14.15 Ermanno Gherardi (UniPv) Welcome and introduction

14.15-14.45 Attilio Rigamonti (UniPv) First NMR Steps Under Luigi Giulotto 1

4.45-15.30 Janez Plavec (Slovenian NMR center) Structural plasticity of G-rich DNA regions

15.30-15.45 Coffee break

15.45-16.15 Rita Puglisi (King’s College London) Joint use of nuclear magnetic resonance and native mass spectrometry in the investigation of protein interactions

16.15-16.45 Giovanna Musco (UniMi) Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment

16.45-17.15 Giorgio Colombo (UniPv) Dynamic disruption of protein interactions

17.15-17.45 Chiara Marabelli (UniPv) Molecular mechanism of nucleosome recognition by the histone demethylases

Day 2 – 14th of September

9.30-10.15 Sasi Conte (King’s College London) Insights into protein-RNA recognition from the La-related protein superfamily

10.15-10.45 Annamaria D’Ursi (UniSa) NMR screening of new potential inhibitors if farnesyl pyrophosphate synthase

10.45-11.00 Coffee break

11.00-11.30 Caterina Alfano (RiMed) Recombinant mussel protein Pvfp-5β: a potential tissue bioadhesive

11.30-12.00 Roberta Spadaccini (UniSannio) MNEI, a sweet protein for every season

12.00-12.30 Andrea Motta (CNR) Novel of the Phenotype

12.30-13.00 Neri Niccolai (UniSi) From PISA to PAVIA: an amazing bioinformatic journey (spiced with some NMR)

13.00-14.00 Lunch

14.00-14.45 Mario Piccioli (UniFi)14.45-15.15 Orlando Crescenzi (UniNa)

15.15-15.30 Coffee break

15.30-16.00 Stefano Mammi (UniPd) Targeting the bcl-2 protein family:  a combined NMR and in-silico approach for fragment based drug discovery

16.00-16.30 Mariagrazia Pizza (GSK) New technologies and vaccines

16.30-17.00 Claudio Toniolo (UniPd) Personal Recollections From A Life-Long Scientific Journey With Pierandrea

17.00-17.10 Concluding Remarks

Presentation Abstracts

First NMR Steps Under Luigi Giulotto
Attilio Rigamonti
University of Pavia

The very first NMR experiments carried out in Pavia since the 1946 will be recalled. The relevant achievements during the years 1947- 1960, such as the development of the method of fast adiabatic passages, the negative spin temperature and some relaxation studies in liquids, will be summarized. A glance to the subsequent studies in the field of the solid state physics will just outlined.


Structural Plasticity Of G-Rich Dna Regions
Janez Plavec
Slovenian NMR center, National Institute of Chemistry, Ljubljana, Slovenia University of Ljubljana, Faculty of Chemistry and Chemical Technology, Slovenia
EN-FIST Centre of Excellence, Trg OF 13, Ljubljana, Slovenia
* e-mail:

DNA can adopt various secondary structures including quadruplexes and i-motifs. The canonical Watson-Crick paired duplexes play a major role in inheritance of genetic material and gene expression. Other structures have been associated with many different biological functions of DNA. The most well studied alternative DNA structures are G-quadruplexes. They are formed by G-rich sequences and consist of four-stranded columnar structures. G-quadruplexes are stabilized by the stacking of multiple Hoogsteen-hydrogen-bonded G-quartets. Notably, cations residing in the center of G-quartets contribute additional stabilizing factor through electrostatic interactions. G-rich sequences have been identified in numerous regions of human genome including chromosomal telomeres and many gene promoters, which play important roles in DNA recombination, replication, transcription, translation, and many other critical biological processes. Discoveries of these sequences have led to significant interest in finding ways to control or modulate formation of G-quadruplexes. Our laboratory has been using NMR extensively to uncover structural details of G-quadruplexes in relation to sequence details, presence of cosolutes, interaction with ligands and even expand the structure and sequence complexity of DNA four-stranded architectures by discovering AGCGA-quadruplexes.

1. A. Kotar, R. Rigo, C. Sissi, J. Plavec, Nucleic Acids Res. 2019, 47, 2641-2653.
2. S. Hadži, V. Kocman, D. Oblak, J. Plavec, J. Lah, Angew. Chem. Int. Ed. 2019, 58, 2387-2391.
3. S. Bielskutė, J. Plavec, P. Podbevšek, J. Am. Chem. Soc. 2019, 141, 2594-2603.
4. S. Takahashi, K. T. Kim, P. Podbevšek, J. Plavec, B. H. Kim, N. Sugimoto, J. Am. Chem. Soc. 2018, 140, 5774-5783.
5. M. Lenarčič Živković, J. Rozman, J. Plavec, Angew. Chem. Int. Ed. 2018, 57, 15395-15399.


Joint use of Nuclear Magnetic Resonance and native Mass Spectrometry in the investigation of protein interactions
Rita Puglisi
King’s College London

The iron-sulfur (FeS) cluster biosynthesis is a process that involves several components. The main ones are a scaffold protein, IscU, on which the FeS cluster is formed and a desulfurase, IscS, which produces sulfur from cysteine. The energy of the process is provided by the binding of a specialized ferredoxin, FdX. CyaY, the bacterial homologue of frataxin, is thought to be the regulator of the cluster formation rate and competes with YfhJ (also called IscX). The cluster is then transferred to other target proteins, through a process that is thought to be mediated by a system of chaperones: HscA (DnaK-like) together with HscB (DnaJ-like).
The function of the machinery for the FeS formation is based on a complex network of interactions. Different approaches as NMR and Native MS have been used synergistically in their investigation. Nuclear Magnetic Resonance (NMR) has been applied to determine the surfaces of binding. With Native mass spectrometry (MS), a technique able to maintain protein structure, folding and interactions, we investigated the species formed during the FeS cluster biosynthesis, to confirm and to extend the network of interactions observed among them.

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment
Giovanna Musco
Biomolecular NMR Unit
Division of Genetics and Cell Biology
c/o Ospedale S. Raffaele

Extracellular High-Mobility Group Box-1 (HMGB1) functions as a cytokine-like molecule that triggers inflammation following infection or injury. It promotes the recruitment of inflammatory cells to injured tissues by forming a heterocomplex with C-X-C Motif Chemokine Ligand 12 (CXCL12) and signaling via its receptor CXCR4. HMGB1 also supports tumorigenesis in inflammation-related malignancies. Thus, it is an important therapeutic target. Here we show that Diflunisal (DFL), an aspirin-like nonsteroidal anti-inflammatory drug, binds to both HMGB1 and CXCL12, and inhibits in vitro and in vivo HMGB1’s chemotactic activity at nanomolar concentrations, at least in part by disrupting the HMGB1-CXCL12 heterocomplex. Our results add new structural/functional insights into the anti-inflammatory activity of DFL, and reveal an unusual mechanism of inhibition, whereby the inhibitor binds to both partner proteins in a heterocomplex.

Dynamic disruption of protein interactions
Giorgio Colombo
Department of Chemistry, University of Pavia, Pavia (Italy)

Here, I will present novel computational strategies to investigate the roles of sequence and structure in determining the interaction properties of proteins with antibodies and chaperones, with the final goal to predict specific antibody or chaperone binding sites, a task that has proven particularly challenging so far.
The antibody binding properties of an antigen depend on its structural dynamics. We have thus developed a method that is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein allowing them to undergo conformational changes, to be recognized by a binding partner and to tolerate mutations with minimal energetic expense. Analyzing the results on isolated proteins and benchmarking against antibody-complexes, the method successfully identifies antibody binding sites.
We test our predictions on antigens from B.pseudomallei, the etiological agent of melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Predicted epitopes are engineered as synthetic peptides and shown to be selectively immunorecognized to the same extent as recombinant proteins in sera from melioidosis-affected subjects. Moreover, antibodies raised against designed sequences prove to be bactericidal.
Next, we focus on the clients interacting with the molecular chaperone Hsp90: starting from the analysis of the TK family of proteins, we develop a model that quantitatively correlates the energetic stabilization profile of each kinase to its “Hsp90-clientness”. We then extend this model to other kinase families, identifying the determinants of folding/unfolding that correlate with the tendency to form complexes with Hsp90. We use this information to design peptide-based kinase-mimics that target Hsp90 and may act as Hsp90-targeted Protein-Protein inhibitors. We will discuss the implication of these methods in vaccine development and in the discovery of new molecular tools with therapeutic applications.

A tail-based mechanism drives nucleosome demethylation by the LSD2/NPAC multimeric complex
Chiara Marabelli
Dept Biology and Biotechnology “Lazzaro Spallanzani”, Univ. of Pavia, Italy

LSD1 and LSD2 are the only known histone demethylases of the flavin class. They share the same substrate H3K4me1/2, but their identical catalytic domains are embedded into two strikingly different protein structures. They are recruited at independent chromatin loci, with strikingly opposite results: LSD1 silences gene promoters whereas LSD2 sustains the elongating RNA polymerase-II at gene bodies. LSD1 stably docks the nucleosome substrate through its corepressor partner CoREST1, whom binding to DNA is essential for nucleosome complex formation. On the contrary, LSD2 lacks any structural appendix for CoREST binding, yet it exposes a unique Zn-finger module of unknown function. We tackled the challenge of describing the molecular mechanism for nucleosome recognition by LSD2. An in-house strategy for the specific crosslinking of flavoenzymes to their nucleosome substrates allowed us to purify the stable LSD2/nucleosome complex for cryo-EM analysis. Our comprehensive approach also employed other mutational and kinetic analysis. Differently from LSD1, LSD2 interaction with the nucleosome does not rely on DNA binding, but on the high-affinity interaction with the forty-aminoacids long H3 N-terminus. Moreover, LSD2 can approach the nucleosome from multiple, equally productive orientations. In the chromatin context, a very short segment (residues 214-225) of the nuclear multi-domain protein NPAC promotes the capture of the H3 N-terminal tail. Further studies on NPAC revealed that its C-terminal dehydrogenase domain was selected during evolution for an inactivating mutation and became a stable tetramerization module. Long flexible linkers extending from NPAC core, hosting the LSD2-binding, connect to an N-terminal chromatin reader domain probably involved in target selection. We hypothesize the multimeric LSD2/NPAC system allows the rapid spreading of H3K4 demethylation in conjunction with the actively transcribing RNA polymerase.

Insights into protein-RNA recognition from the La-related protein superfamily
Sasi Conte
King’s College London

RNA binding proteins (RBPs) regulate gene expression post-transcriptionally and alterations in their function result in aberrant expression profiles and disease. Yet, how exactly RBPs are able to select and bind to their RNA targets remains unknown. Here, our findings on several RBPs belonging to the superfamily of La-related proteins (LARPs) will be presented, revealing unexpected and exciting features of protein-RNA recognition. Particularly interesting are the latest investigations on LARP4A, revealing that RNA binding is mediated by disordered regions that do not contain recognisable RNA binding motifs.

NMR screening of new potential inhibitors of farnesyl pyrophosphate synthase
Anna Maria D’Ursi
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084, Fisciano (Salerno), Italy.

N6-isopentenyladenosine (i6A), a modified nucleoside belonging to the cytokinin family, shows many in vitro and in vivo biological actions, including antitumoral effects and inhibition of farnesyl pyrophosphate synthase (FPPS) activity. FPPS catalyzes the synthesis of isoprenoids and plays a key role in many cancer-related pathways; therefore, it is considered a promising target for new anti-cancer therapies.
Structural interaction between i6A and FPPS was predicted by inverse virtual screening approach and confirmed by ligand-based NMR interaction. Starting from the structural requirements dictated by i6A binding mode in the active site of FPPS enzyme, new i6A derivatives were designed, synthesized, and tested using NMR screening and antitumoral biological assays. N6-benzyladenosine derivatives were identified as a new molecular tool characterized by improved FPPS inhibitory activity, and exerting combined cytotoxic and immunostimulatory activities.

Recombinant mussel protein Pvfp-5β: a potential tissue bioadhesive
Caterina Alfano
Fondazione Ri.MED, Palermo, Italy

Many marine organisms rely on natural adhesives to attach to various surfaces under wet conditions for their life-cycle, movement and self-defense in aqueous tidal environments. Mussel adhesive proteins have received increased attention in recent years for their potential applications in several fields, such as medicine, biomaterials and biotechnology being biocompatible and able to elicit minimal immune response. The Asian green mussel Perna viridis secretes several byssal plaque proteins. The Perna viridis foot protein-5β (Pvfp-5β) is the first protein to initiate interaction with the substrate, displacing interfacial water molecules before binding to the surface. Here, we present a study of recombinant Pvfp-5β, in which we established the first recombinant expression in E. coli of the protein. We characterized the protein showing that, despite the circular dichroism spectrum with features of a random coil, the protein is correctly folded as demonstrated by mass spectrometry and nuclear magnetic resonance. We evaluated the cell viability and cell adhesion capacity of Pvfp-5β using NIH-3T3 and HeLa cell lines. Our results revealed that the protein has no cytotoxic effect at the investigated protein concentrations and a good cell adhesion strength on both glass and plastic plates. Overall, we show that the adhesive properties of recombinant Pvfp-5β make it an efficient surface coating material, suitable for biomedical applications including regeneration of damaged tissues.

MNEI a protein for every season
R. Spadaccini
Dipartimento di Scienze e tecnologie-Universita’ degli studi del Sannio-Benevento Italy

Sweet proteins are a family of a few unrelated members isolated from the fruits and seeds of some tropical plants. As they show a sweetening power up to five orders of magnitude greater than sucrose, they are particularly attractive as potential scaffolds for the design of the next generation of sweeteners. In our laboratory, we have been focusing on the study of the sweet protein monellin and its single chain derivative MNEI, provided with superior thermal stability.
During my presentation I will give an overview of the work on MNEI, starting from the early work done in Temussi’s lab on MNEI structure and mutant design to more recent work on the aggregation properties of MNE

Novel of the phenotype
Andrea Motta
CNR – Naples

The seminar will describe the relevance of the phenotype in personalized medicine. The metabolomics approach will be briefly introduced by resorting to two examples that illustrate how phenotypes (endotypes and metabotypes) can be determined by using NMR spectroscopy and statistical analysis.
Few digressions on Gottfried Benn novel (Roman des Phänotyp, 1949) and early Temussi life will also be presented.


From PISA to PAVIA: an amazing bioinformatic journey (spiced with some NMR)
Neri Niccolai
University of Siena & Le Ricerche del BarLume

Understanding the language that proteins use to call their molecular partners, is a fundamental step to decipher Life at atomic resolution. Protein Data Bank contains an unprecedented array of pictures with proteins in the act of talking with other proteins, nucleic acids, ions and small molecules. By assuming that amino acid occurrences at protein interfaces with such a large variety of ligands hide signals of protein language, through structural bioinformatic investigations, we have shown how Arg/Lys ratio determines protein sliding speed along DNA rails (1). With the same approach, we have established the role of Gly in shaping enzyme active sites, opening new perspectives to protein engineering and Molecular Medicine (2).

1) Gardini S, Furini S, Santucci A, Niccolai N. A structural bioinformatics investigation on protein-DNA complexes delineates their modes of interaction. Mol Biosyst. 2017 May2;13(5):1010-1017. doi: 10.1039/c7mb00071e.
2) Niccolai N, Bongini P, Trezza A, Mangiavacchi G, Santucci A, Spiga O, Bianchini M, Gardini S Structural bioinformatic survey of protein-small molecule interfaces delineates the role of glycine in enzyme active sites. BBA, 2019 submitted.

Are Nmr Structures In Solution Really Boring?
M. Piccioli
Magnetic Resonance Center and Department of Chemistry, University of Florence, Italy
The golden age of NMR solution structures of biomolecules has left a legacy of established sets of experiments and automated and semi-automated approaches for resonance assignments and structure calculation. However, there are still many cases where NMR structures (and NMR characterization as well) cannot be performed routinely, thus providing challenging opportunities to develop non-conventional, fully manual and time consuming approaches. A small paramagnetic metalloprotein, where the complete assignment is prevented by paramagnetic relaxation enhancements and where NOEs are sparse due to the combination of small rotational correlation times and large relaxation rates, is indeed one of those cases.
The solution structure of PioC, a 54 aminoacid High Potential Iron Protein (HiPIP) containing a 4Fe-4S cluster isolated from Rh. Palustris, requires a combination of classical structural constraints, based on dipolar couplings, and of relaxation based constraints. In the case of signals that are severely affected by the hyperfine interaction, we need to design novel pulse sequences to measure R2 and R1 rates of resonances that escape detection in conventional experiments. The complete resonance assignment can be performed only by a combination of tailored experiments based on both 1H and 13C direct detection.

Targeting the bcl-2 protein family: a combined NMR and in-silico approach for fragment based drug discovery
Stefano Mammi
University of Padova

Bcl-XL is a member of the Bcl-2 protein family, which plays a fundamental role in the regulation of apoptosis. Bcl-XL plays an anti-apoptotic role physiologically but its overexpression in tumor cells represses cellular death and this is associated with tumorigenesis. Inhibitors of these proteins re-establish apoptosis pathways balance and cause tumor regression.
Fragment Based Drug Discovery (FBDD) is a very efficient method to obtain high affinity ligands. Low molecular weight molecules, typically with low affinity, are identified and chemically combined to obtain stronger ones. NMR is the technique of choice for this task because of its ability to detect weak binding, especially when paralleled by in silico approaches. While NMR can exploit computations to improve its throughput, no computational approach uses NMR data to narrow down its search. The goal of this work is to fill this gap with the definition of an experimental-NMR-based Molecular Dynamics protocol. The search for Bcl-XL inhibitors is a perfect application for this procedure.

New Technologies and vaccines
Mariagrazia Pizza
GSK Vaccines, Siena, Italy

Vaccines have had a major impact on human health over the past two centuries, allowing for the control and elimination of many infectious diseases. Most of the vaccines available today, although very effective, have been developed using conventional approaches. Important discoveries in the field of chemistry, microbiology and immunology and the development of new and sophisticated technologies have provided alternatives to the design of improved vaccines or of novel vaccines against infections for which preventive measures do not exist.
The “structural vaccinology” approach, aimed to guide the rational design of ideal vaccine antigens for future vaccines, will be presented in detail.


Personal Recollections From A Life-Long Scientific Journey With Pierandrea
Claudio Toniolo
University of Padova
My talk is essentially focused on two topics: i) my publications co-authored with great experts of the pioneering years of NMR, and ii) my publications with Pierandrea Temussi on amino acid/ peptide/ protein sweeteners.



Efstathios Charlaftis
Riaz Hussain
Pietro Carretta
Mariangela Jessica Alfeo
Mattia Moiola
Federico Forneris
Maira Rossi
Francesca Bastaroli
Caterina Temporini
Andrea Alfieri
Federica De Leo
Federica Corana
Francesco Bonomi
Giuliana Marsico
Gennaro Guarino
Massimo Serra
Mario Piccioli
Stefano Mammi
Claudio Toniolo
Sasi Conte
Caterina Alfano
sandro conticelli
Giuliana Marsico
Andrea Motta
Neri Niccolai
Stefania Iametti
Andrea Motta
Serena Faggiano
Samantha Gonah
Roberta Spadaccini
Guido Tiana
Fabio Temussi
Ermanno Gherardi
Teresa Recca
Francesca Vasile
giovanna musco
Rigamonti attilio
Rita Puglisi
Angela Bitonti
Monica Passananti
Patrizia Arcidiaco
Claudia Scotti
Andrea Marsala
Luisa Iamele
Mariella Mella
Elena Poggio
Maria Antonietta Castiglione morelli
Francesca Ferrari


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