Upload
lydung
View
217
Download
0
Embed Size (px)
Citation preview
Scientific Annual Report 2011
2
Acknowledgements
We thank the following institutions for their stimulating interaction:
ProtEra Srl C’era fino a meta’ 2011!!!
Fiorgen Onlus
Boehringer Spa
Bracco Spa
Bruker Bio Spin Srl
Novartis Vaccines
Siena Biotech
Menarini
Scientific Annual Report 2011
3
Introduction
WHO WE ARE
The CERM, Center for Magnetic Resonance, is a scientific institution for research and higher education
at the University of Florence. It is also the national laboratory for the Interuniversity Consortium for
Magnetic Resonance of Paramagnetic Metalloproteins (CIRMMP) which embraces three important
Universities: Florence, Siena and Bologna. CERM is an infrastructure for structural biology with a
particular focus on Life Sciences and specializations in bioinformatics, molecular biology, structural
biology, novel drug design and development and metabolomics.
Being an important laboratory at both national and international level, CERM receives funding from both
the Italian Ministry of Higher Education and Research and the European Commission. Since 1994
CERM has been recognized as a transnational research infrastructure for its expertise and state-of-the-
art instrumentation.
The principal technology used at CERM is NMR spectroscopy and the onsite instrumentation is among
the most advanced in the world. A European transnational access service financed by the EC since
1994 in addition to a national service operating since 1990 places CERM at the top of the list in NMR
networking. Its two-fold status as a public-private laboratory fosters interaction between private industry
and public research institutions such as University and CNR and European counterparts, promoting
synergetic activities such as collaborations and services to SMEs.
CERM is involved in the ESFRI (European Strategy Forum on Research Infrastructures) and Italian
roadmaps and it is one of the 7 “core centers” of the European project for the establishment of an
Integrated Structural Biology Infrastructure (INSTRUCT). CERM is an e-infrastructure managing a GRID-
based platform with other laboratories in Europe, for biomolecular structure calculation (WeNMR -
www.wenmr.eu), funded by the European Commission. It also promoted the creation of a biobank of
samples of biological and biomolecular resources Da Vinci European Biobank
(https://www.davincieuropeanbiobank.org/home.php).
WHERE WE ARE
CERM is located in the Scientific Campus (“Polo Scientifico”) of the University of Florence in Sesto
Fiorentino, an area just west of Florence, Italy. The main building has over 3000 square meters of
usable space and contiguous laboratories measure more than 500 square meters. The campus borders
Florence International Airport and is a mere 15 minutes from the world-renowned center of renaissance
art and culture, Firenze.
Scientific Annual Report 2011
4
WHAT CERM IS (http://www.cerm.unifi.it)
A Center for Research, Technological Transfer and Higher Education at the University of Florence
A National Laboratory for the Interuniversity Consortium CIRMMP
managing national access since 1990
managing transnational European access since 1994
A Core center in the European Integrated Structural Biology Infrastructure (INSTRUCT
www.structuralbiology.eu) An e-infrastructure managing a GRID service for computation in structural biology (www.wenmr.eu).
A Host for the Da Vinci European Biobank (https://www.davincieuropeanbiobank.org/home.php)
WHAT CERM DOES
Highlights of research at CERM:
The related activity of CERM can be summarized in the scheme (see next page). During 2011 a number
of projects have been developed, either as an extension of the activities of previous years or as new
projects: research, training, access to infrastructure. Most of these projects receive specific funding from
national and European organizations.
Investments in 2011 amount to € 2.055.000 in research: € 570.000 towards training and education, €
965.000 towards equipment and € 520.000 towards research activities. An additional € 360.000 went for
operational expenses.
The value of instrumentation on location at CERM is close to € 40.000.000.
Our 2011 student body includes 29 PhD students, 24 postdoctoral scientists and 6 graduate and
undergraduate students.
CERM has provided instrumentation access to several research groups and has managed national
access funded by MIUR. The interdisciplinary character of CERM research projects combined with the
excellence of its instrumentation symbolize a point of reference for the scientific community and for
cultural growth in the country, as demonstrated by increased usage on the part of national scientists.
Scientific Annual Report 2011
6
Table of Contents
Financers ……………………1
Acknowledgments ……………………2
Introduction ……………………3
Who we are ……………………3
Where we are ……………………3
What CERM is ……………………4
What CERM does ……………………4
Table of contents ……………………6
CERM organization ……………………7
Staff ……………………8
Research areas ……………………12
Structural biology ……………………12
NMR methodology ……………………13
Metabolomics ……………………14
Bioinformatics and databases ………………15
GRID ……………………16
In-cell NMR and Systems Biology………….. 17
Relaxometry ……………………18
X-ray crystallography ……………………19
Drug discovery ……………………21
International doctorate ……………………22
European NMR service ……………………23
NMR National service ……………………24
Collaborative research ……………………25
NOVARTIS VACCINES ……………………25
FIORGEN ……………………26
PROTERA ……………………27
Protein structures 2011 ……………………28
Meetings and Seminars ……………………32
Publications ……………………34
Cover image: PDB code: 2KC0
Scientific Annual Report 2011
7
CERM- CIRMMP organization
SCIENTIFIC BOARD OF CERM UNIVERSITY Florence
Prof. Ivano Bertini , Director
Prof. Lucia Banci
Prof. Claudio Luchinat
Prof Roberta Pierattelli
Prof. Antonio Rosato
Prof. Paola Turano
CIRMMP BOARD OF DIRECTORS Florence
Prof. Ivano Bertini , Director
Prof. Claudio Luchinat
Prof. Lucia Banci
Bologna
Prof. Stefano Ciurli
Prof. Francesco Capozzi
Siena
Prof. Elena Gaggelli
Prof. Stefano Mangani
CIRMMP SCIENTIFIC BOARD Florence
Prof. Lucia Banci
Prof. Ivano Bertini
Prof. Claudio Luchinat
Siena
Prof. Gianni Valensin
Utrecht
Prof. Rolf Boelens
Frankfurt
Prof. Harald Schwalbe
PANEL FOR NATIONAL ACCESS Prof. Henriette Molinari - Verona
Prof. Lucia Zetta - Milan
Scientific Annual Report 2011
9
FACULTY AND RESEARCHERS
University of Florence
Department of Chemistry
Dr. Claudia Andreini
Prof. Ivano Bertini
Prof. Lucia Banci
Dr. Francesca Cantini
Prof. Simone Ciofi-Baffoni
Prof. Isabella Felli
Dr. Marco Fragai
Prof. Claudio Luchinat
Prof. Cristina Nativi
Prof. Giacomo Parigi
Prof. Mario Piccioli
Prof. Roberta Pierattelli
Prof. Antonio Rosato
Prof. Paola Turano
University of Siena
Department of Chemistry
Prof. Stefano Mangani
Prof. Elena Gaggelli
Prof. Gianni Valensin
University of Bologna
Department of Agricultural and Environmental Sciences
Prof. Stefano Ciurli
Dr. Barbara Zambelli
Department of Food Sciences
Prof. Francesco Capozzi
Dr. Elena Babini
Scientific Annual Report 2011
10
POST DOCTORAL SCIENTISTS
Valentina Borsi
Vito Calderone
Gabriele Cavallaro
Chiara Cefaro
Leonardo Decaria
Karolina Gajda
Angelo Gallo
Eva Grassi
Xiaoyu Hu
Tatiana Kozyreva
Daniela Lalli
Farrell Ian MacKenzie
Maxime Melikian
Manuele Migliardi
Maciej Mikolajczyk
Francesco Musiani
Antonella Nesi
Anna Pavelkova
Gianfranco Picone
Elena Porciatti
Cecilia Pozzi
Jeffrey T. Rubino
Chiara Venturi
Miguela Vieru
Massimiliano Vignali
Julia Winkelmann
PHD STUDENTS
Letizia Barbieri
Olga Blazevits
Stefano Cacciatore
Edoardo Calçada
Chiara Calugi
Linda Cerofolini
Lucio Ferella
Deepa Jaiswal
Vasantha Kumar
Serena Lorenzini
Enrico Luchinat
Jiafei Mao
Kathleen McGreevy
Malini Nagulapalli
Sara Neri
Riccardo Peruzzini
Enrico Ravera
Mauro Rinaldelli
Camilla Rosa
Vaishali Sharma
Mirco Toccafondi
Scientific Annual Report 2011
11
GRADUATE STUDENTS
Sara Fanciulletti
Gianluca Gallo
Thomas Hosek
Luca Mazzei
Martina Norcini
Claudia Oteri
Corinne Portioli
VISITING STUDENTS Joao Teixeira
TECHNICIANS (UNIVERSITY AND CIRMMP)
Marco Allegrozzi
Fabio Calogiuri
Rebecca Del Conte
Andrea Giachetti
Leonardo Gonnelli
Massimo Lucci
Cristina Mescalchin
Enrico Morelli
ADMINISTRATIVE SUPPORT (UNIVERSITY AND CIRMMP)
Annalisa Cauteruccio
Francesca Di Gloria
Simona Fedi
Milena Moazzi
Francesca Morelli
Laura Norfini
Lisa Orlando
Scientific Annual Report 2011
12
Research Areas STRUCTURAL BIOLOGY
Structural biology is a branch of molecular biology, biochemistry, and biophysics concerned with
the molecular structure of biological macromolecules, especially proteins and nucleic acids, how
they acquire the structures they have, and how alterations in their structures affect their function.
This subject is of great scientific interest because macromolecules carry out most of the functions
of cells, and because it is only by folding into specific three-dimensional shapes that they are able
to perform these functions.
Regarding Structural Biology CERM is currently studying some classes of metalloproteins involved
in the homeostasis of metals. Each organism possesses mechanisms of homeostatic regulation
that allow the uptake of the quantity of metal necessary for the cell while preventing accumulation
over toxic levels. A category of proteins, called metallochaperones, is responsible for the transpor-
tation of metals in cells and their distribution to target enzymes or to ATPases that allow them to
pass through membranes.
We have characterized the structure, dynamics and protein-protein interactions of some of these
systems in eukaryotes and prokaryotes. The principal metals we studied are: Cu, Ni and Fe.
Natural mutations in some of these proteins lead to disease. We study the biophysical and struc-
tural characteristics of some mutants to understand their relationship with disease.
Among the mutants we have studied is the human copper protein zinc superoxide dismutase
(SOD1). Mutations in the SOD1 gene have been associated with some familial forms of a neuro-
degenerative disease of the motor neurons called Amyotrophic Lateral Sclerosis (ALS).
Metallochaperones have also been identified in the mitochondrion, where they have a role in the
formation of iron-sulphur clusters and in the assembly of copper enzymes such as cytochrome c
oxidase and SOD. Some proteins involved in these mechanisms have been structurally character-
ized and their interactions have been studied at the molecular level. We have identified a redox
protein dependent machinery involving a disulfide relay system that determines the folding of a
class of mitochondrial proteins. This disulfide ex-
change reaction between the oxidase Mia40,
which is the first protein involved in the oxidative
folding pathway, and its substrates Cox17 and
Tim10, has been characterized at the molecular
level. The second step of the oxidative folding
pathway has been also characterized at the mo-
lecular level describing the interaction and elec-
tron transfer mechanisms between Mia40 and its
physiological electron acceptor, the ALR protein.
Recently, we have found that this mechanism is
involved in the biogenesis of iron-sulfur cluster
proteins. In particular, we found that a [2Fe-2S]
cluster-containing protein named anamorsin is a
Mia40 substrate. The structure of anamorsin and
its interaction with Mia40 have been character-
ized for the first time at the molecular level. The
functional role of this important protein is now
under investigation through the characterization
of its interactions with their protein partners.
The hydrophobicity-driven substrate mimicry mechanism operating in the oxidative protein trapping of mitochondrial intermembrane space (IMS)-imported proteins. Mia40 oxidoreductase is in red, the IMS-imported substrate Cox17 is in gray and the sulfhydryl oxidase ALR is in dark green. The interacting hydrophobic regions of these proteins are green and the cysteines in-volved in the disulfide exchange reactions are in yellow.
Scientific Annual Report 2011
13
NMR METHODOLOGY
The development of new NMR methods is essential for broadening the range of NMR spectros-
copy applications in the study of different biological systems.
Recently, focus has been placed on 13C direct detection NMR spectroscopy both in solution and
solid state. A suite of multidimensional NMR experiments based on 13C detection has been devel-
oped and offers an additional tool for the study of biological macromolecules that can provide
more, and in some cases unique, information particularly for molecules not easily studied by other
NMR techniques, such as intrinsically disordered proteins.
The most recent developments focuses in the implementation of the so called “fast methods” for
the study of these proteins directly in-cell, both bacterial and eukaryotic. As the experiments based
on 13C detection in solution shares some common aspects to experiments in the solid state, some
of the innovations introduced in solution NMR were exploited to optimize the performances of sev-
eral solid state NMR experiments. The key development in solid state NMR pursued lately, how-
ever, resides in the possibility to use 1H NMR experiments, enabled by the use of high-MAS pro-
beheads at high magnetic field.
The figure reports Strips from the
proton-detected (H)NHHRFDR ex-
periment (3.3 ms mixing time). The
diagonal peak in each strip is cir-
cled, whilst solid lines indicate trivi-
al contacts (|i−j|=1) which were not
used in the structure calculation.
The long-range contacts detected
in a) are depicted on the crystal
structure of the CuII,Zn
II-SOD hom-
ologue.
Furthermore, recently, Sedimented Solutes NMR (SED-NMR) has been developed as a simple and
minimally perturbing method to prepare biological samples for solid state NMR. SED-NMR is based
on the limitation of rotational diffusion of biomolecules due to self-crowding induced by ultracentrif-
ugation in the MAS or in a real ultracentrifuge with a suitable device. This methodology can be ap-
plied on proteins above 20 kDa. It might be a suitable methodology to observe transient complexes
or oligomeric states that fail to crystallize and/or are too big to be observed by routine methods of
solution state NMR.
Spectra are usually of high resolution, even higher than the corresponding microcrystalline prepa-
ration. Published results are available on Ferritin, Bovine Serum Albumin and Carbonic Anhydrase
II.
Scientific Annual Report 2011
14
METABOLOMICS
Metabolomics is a further “omic” science that is now emerging with the purpose of elaborating a
comprehensive analysis of the metabolome, which is the complete set of metabolites (i.e. small
molecules intermediates) in an organism, tissue, cell or biofluid. While genomics tells you what
could happen, metabolomics tells you what is really happening. There are close to hundred thou-
sand proteins encoded in the genome, but only a few thousand metabolites. The projects involve
varied areas of interest, in particular the biomedical field. The primary current metabolomic pro-
jects, are:
Celiac disease, potential celiac disease and gluten sensitivity
Broncho-pulmonary obstructive diseases using exhaled breath condensate
Study on metabolic fingerprint of heart failure
New statistical methods for data analysis and clustering
Prognostic studies on patients with metastatic colorectal cancer
Analysis of saliva samples for periodontal diseases fingerprinting
The use of metabolomics for developing better standard operating procedures for samples
treatment ad storage
Discovery of new biomarker for diagnosis and prognosis of melanoma
Metabolomics application to urogenital cancers (prostate, bladder, kidney)
Metabolomics of obesity and bariatric surgery
The study of these fluids is performed through
nuclear magnetic resonance (NMR) spectroscopy.
Data obtained from NMR spectra are analysed
with advanced computational techniques such as
supervisioned learning methods (Support Vector
Machines, Neural Networks) and with standard
techniques such as of primary components anal-
yses or multilinear regression methods. The goal
of metabolomics is to integrate the individualiza-
tion specific metabolomic markers with the set of
cellular products to explain the working inner
workings of the onset of disease.
Reconstruction one-dimensional 1H NMR spectra of urine by back-projection into the original space of their representation in the 21-dimensional discriminating subspace. (a) Image of the original bucket ta-ble. (b) Image of the table reconstructed from the bucket spectra represen-tation in the 21-dimensional donor-discriminating PCA/CA subspace. (c) Median reconstructed spectra for individual donors (blue, male; red, female).
Scientific Annual Report 2011
15
BIOINFORMATICS AND DATABASES
In 2011, the research activity in bioinformatics at CERM was carried out, like in previous years,
within the general framework of the study of metalloproteins, following two main thematic lines
concerning (i) the development of databases, and (ii) the comparative analysis of metalloproteins’
sequences and structures. These two lines are pursued in a strongly integrated fashion, since the
structuring of information into databases facilitates the development of the methods of analysis for
the systems of interest, and, on the other hand, the results of the analyses are useful to optimize
the architecture and features of the databases.As regards point (i), we mainly worked on the
construction of a new public database called MetalPDB, whose aim is to organize the current
knowledge on metalloproteins in the most effective way for the scientific community, integrating the
structural information contained in the PDB with the functional information available in the
literature. This project is funded in the framework of the FIRB program “Futuro in ricerca”.
Furthermore, we worked on the maintenance of Metal-MACiE (http://www.ebi.ac.uk/thornton-
srv/databases/Metal_MACiE/home.html), a public database that contains information on the
properties and functions of metals in metalloenzymes, and was developed in collaboration with the
European Bioinformatics Institute (EBI). As regards point (Ii), we carried out structure-function
relationship studies on some classes of proteins of particular interest for our laboratory, specifically
(a) zinc proteins, (b) zinc enzymes, (c) a family of copper proteins called Sco, and (d) c-type
cytochromes in complex with other proteins. In the case of zinc proteins, we were able to classify
77% of zinc sites found in proteins into ten clusters, each grouping zinc sites with structures that
are highly similar, and an additional 16% into seven pseudo-clusters, each grouping zinc sites with
structures that are only broadly similar. Sites where zinc plays a structural role are predominant in
eight clusters and in two pseudo-clusters, while sites where zinc plays a catalytic role are
predominant in two clusters and in five pseudo-clusters. In the case of zinc enzymes, we estimated
that 10% of the chemical reactions catalyzed by enzymes involve at least one catalytic mechanism
that requires zinc to occur. Zinc ions playing a catalytic role are typically used in substrate binding
and activation, although their mechanism of action varies to some extent between hydrolases and
the other enzyme classes. Hydrolases are the most common zinc enzymes in all domains of life,
and are more widespread in prokaryotes than in eukaryotes. In the case of Sco proteins, we
hypothesized that during evolution Sco proteins, starting from a primary role as thiol-disulfide
oxidoreductases associated with a conserved CXXXC motif, acquired copper-binding properties by
way of specific mutations that occurred in the surroundings of the CXXXC site. Specifically, a
histidine ligand in a flexible loop region close to that site appears to be the main factor that
modulates the metal affinity of the protein, bringing about specific combinations of redox and
copper-binding properties which ultimately determine the functional versatility of Sco proteins. In
the case of c-type cytochromes, finally, we observed that certain physico-chemical properties are
statistically different in transient adducts with respect to complexes that are permanent or are found
in multi-domain proteins. These properties include the extent of the protein interface area, the
amino acid composition and the packing density at the interface. In addition, we identified three
different structural modes of interaction by which we were able to classify all known structures
(except one) of complexes involving cytochrome c.
Scientific Annual Report 2011
16
GRID
CERM is a node of the electronic infrastructure WeNMR (www.wenmr.eu). This is an international
project funded by the European Commission that aims to expand the services created by the pre-
vious e-NMR project. The overall goal is to enable analysis of NMR data, alone or in combination
with other techniques for structural investigation of biomacromolecules (mainly SAXS), in a highly
automated manner using standardized protocols through web portals. The computational infra-
structure that makes this possible is based on grid computing. However, thanks to the use of web
interfaces, the users of WeNMR do not need to come into contact with the grid environment, which
is technologically quite complex. The design is indeed similar to that applied by other services,
technically and informatically much simpler, in use by the biological and biochemical scientific
communities. Within the WeNMR infrastructure, CERM has implemented and maintains the follow-
ing programs: AnisoFit for the fitting of orientation and paramagnetic tensors; Antechamber for the
calculation of force field parameters for organic ligands, aimed at the subsequent use with AMBER
(see below); XPLOR-NIH for the calculation of 3D structures of biomacromolecules from NMR da-
ta; AMBER for structural refinement as well as long molecular dynamics simulations; MaxOcc for
the calculation of the maximum possible occurrence of protein conformations in multi-domain pro-
teins from a combination of NMR data of paramagnetic and SAXS.
Moreover, CERM is coordinating an international initiative to compare the performance of existing
programs for the automatic calculation of protein structures based on experimental data (Critical
Assessment of Automated Structure Determination of Proteins from NMR data - CASD-NMR). The
competition is based on using software to automatically calculate NMR structures from experi-
mental data not yet publicly available (www.wenmr.eu/wenmr/CASD-NMR).
Finally, the CERM is a member of the Italian Grid Initiative, through which is involved in the Euro-
pean Grid Initiative.
Scientific Annual Report 2011
17
IN CELL NMR AND SYSTEMS BIOLOGY
Structural studies of proteins are typically carried out in vitro, far from the physiological conditions of the cellular context. However, proteins and other biomolecules should ideally be characterized within the cellular milieu, taking into account the properties of their physiological environment, in-cluding pH, redox potential, viscosity, and the presence of all relevant interaction partners. In-cell NMR is an emerging technique, which has the ability to acquire structural and conforma-tional information of biomolecules in the native environment of living cells, with atomic resolution. This ability places in-cell NMR in a unique position, combining a structural biology technique with the full complexity of the cellular environment, in a true systems biology approach. In-cell NMR is being applied at CERM to study protein folding and maturation events. Proteins are overexpressed in E. coli cells, which provide a good model to study the influence of the cytoplas-mic environment on protein folding and maturation. The technique is also being extended to human cells. NMR on human cells allows studying human proteins in their real environment, together with their specific partners. This advancement requires careful optimization of many technical aspects, such as tuning cell growth conditions and sample preparation, improving DNA transfection efficiency and maximizing protein overexpression level. The maturation steps of the human protein superoxide dismutase 1 (SOD1) were characterized in living E. coli cells. In order to reach its active conformation, SOD1 has to undergo several post-translational modifications, involving transition from a partially unfolded state to a rigid homodimeric structure, binding of copper and zinc ions and formation of an intramolecular disulfide bond. In ab-sence of metal ions, SOD1 in the cytoplasm is found in the reduced, partially unfolded state. Upon zinc addition, SOD1 dimerizes and binds one zinc ion per subunit in a selective manner, which is only possible in the cellular environment and does not occur in vitro.
The complete sequence of events which lead to the mature, active Cu,Zn-SOD1 form is now being followed in the cytoplasm of human cells. Additionally, the essential role of the copper chaperone for SOD1 in the late maturation steps of SOD1 is being assessed, by co-expressing both proteins in human cells.
In absence of metal ions, human SOD1 in E. coli cells is in the apo form, partially unfolded and reduced. When zinc is added in excess to the external medium, it enters the cells. SOD1 dimerizes and binds only one zinc ion per subunit, with a selectivity which is not observed in vitro.
Scientific Annual Report 2011
18
RELAXOMETRY
Fast field cycling relaxometry is a tool for measuring nuclear relaxation rates from very low magnet-
ic fields (0.01 MHz proton Larmor frequency) up to 1 T (about 45 MHz proton Larmor frequency).
The field dependence of the relaxation rates provides information on the structural and dynamic
features of a molecule (and, in the case of paramagnetic systems, on the electron relaxation).
In the context of the characterization of nitroxide radicals for solution dynamic nuclear polarization
(DNP) experiments, we have collected NMRD (Nuclear Magnetic Relaxation Dispersion) profiles of
solvent water protons in the presence of Frémy’s salt or of TEMPONE. The fit of the relaxation pro-
files using a model for dipolar inner-sphere and outer-sphere relaxation provided the structural and
dynamic parameters needed for determining the coupling factor, on which the solvent DNP en-
hancement depends. NMRD profiles proved also useful for the characterization of possible contrast
agents for MRI. Novel gadolinium(III) complexes, developed using click chemistry, have been pro-
posed as contrast agent with improved efficiency. In optimizing the relaxivity of this new complex
we undertook a study of the linker length between a azide bearing Gd(III) chelate and a trialkyne
scaffold. The complex shows a tenfold increase in the rotational correlation time from 0.049 ns to
0.60 ns at 310 K with respect to the Gd(III) chelate. This determines a 500% increase in relaxivity
at 1.41 T versus small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.
Proton relaxivity of aqueous solutions containing 15N labeled Frémy’s salt depending on the nu-
clear Larmor frequency as measured in a field cycling relaxometer at 298 K, together with the best
fit line. The term resulting solely from I dispersion is shown as dashed line. From this profile a
coupling factor of 0.34 can be estimated.
Scientific Annual Report 2011
19
X-RAY CRYSTALLOGRAPHY
X-ray crystallography is essentially a form of very high resolution microscopy. It enables us to visu-
alize protein structures at the atomic level and enhances our understanding of protein function.
Specifically we can study how proteins interact with other molecules, how they undergo conforma-
tional changes, and how they perform catalysis in the case of enzymes. Armed with this infor-
mation we can design novel drugs that target a particular protein, or understand the role played by
a protein in the frame of cellular metabolism or rationally engineer an enzyme for a specific indus-
trial process.
Crystal structures of proteins began to be solved in the late 1950s, beginning with the structure of
sperm whale myoglobin by Max Perutz and Sir John Cowdery Kendrew. Since that success, over
70000 X-ray crystal structures of proteins, nucleic acids and other biological molecules have been
determined. Moreover, crystallography can solve structures of arbitrarily large molecules. X-ray
crystallography is now used routinely to determine how a pharmaceutical drug interacts with its
protein target and what changes might improve it.
During 2011, 12 new protein crystal structures were solved and 8 of them were deposited and re-
leased in the Protein Data Bank while 4 of them are on-hold waiting for the relevant paper publica-
tion.
Matrix metalloproteinases (MMPs) are a large class of strictly-related zinc-dependent enzymes
which belong to the family of proteolytic enzymes. MMPs are involved in many aspects of physio-
logical cellular processes, as well as in pathologies, such as reumathoid arthritis, pulmonary em-
physema, tumor growth and metastasis. Since many of these pathologies may benefit from the
control of MMPs activity several MMPs inhibitors have been designed and tested in clinical trials.
However, most of them, are poorly soluble in water and suffer from a modest oral bioavailability. A
well-balanced hydrophilic/lipophilic character is crucial for reaching and maintaining high drug lev-
els in plasma. The challenge is therefore that of determining new selective inhibitors of the prote-
ase activity. Structures of MMP-12 complexes were determined with new substrates generated
from in-silico screening (PDB codes: PDB 3RTS, 3RTT, 3RTU). The structure of MMP1 has also
been determined by X-ray crystallography as well as by NMR so that it has been possible to im-
plement a crystallographic refinement software with the use of NMR restraints. Furthermore the
structure of MMP3 in complex with a platinum-based inhibitor has also been determined. Onen of
the relevant papers has been published (Bertini I, Calderone V, Cerofolini L, Fragai M, Geraldes
CF, Hermann P, Luchinat C, Parigi G, Teixeira JM. (2012). The catalytic domain of MMP-1 studied
through tagged lanthanides. FEBS Lett. 586(5):557-67) while two others have been submitted.
Class D beta-lactamases are a heterogeneous group of active-site serine beta-lactamases that
confer resistance to beta-lactam antibiotics, the most widely used family of anti-infective agents, by
hydrolyzing the amide bond of the beta-lactam ring and thus bear significant clinical relevance. For
this reason the study of the interactions between these proteins and ligands can shed light on the
Scientific Annual Report 2011
20
fine tuning of their catalytic function thus offering new structural basis to investigate the potential of
new scaffolds of beta-lactamase inhibitors. The crystal structures of BJP-1 and TEM72 have been
solved and their coordinates depositited under the following PDB codes: 3P98, 3LVZ, 3M8T. The
relevant paper has been published (Docquier JD, Benvenuti M, Calderone V, Rossolini GM,
Mangani S. (2011). Structure of the extended-spectrum -lactamase TEM-72 inhibited by citrate.
Acta Crystallogr Sect F Struct Biol Cryst Commun; 67(Pt 3):303-6).
Several enzymes are known to catalyze dithiol-disulfide transfer reactions between proteins to effi-
ciently form or disrupt disulfide bonds. A class of them includes sulfhydryl oxidases that are capa-
ble of forming disulfide bonds de novo. In general, these enzymes exist as homodimers, depend on
FAD as a cofactor, and use oxygen as final electron acceptor. They typically contain a CXXC motif,
involved in the redox-reaction and close to the FAD molecule. ALR (augmenter of liver regenera-
tion) is a sulfhydryl oxidases working in the intermembrane space of mitochondria. ALR is found in
a large number of different cell-types and tissues. Its activity is essential for the survival of the cell,
for the biogenesis of mitochondria and for the maturation of cytoplasmic proteins with
mitochondrially assembled iron–sulfur clusters. ALR is found in two main alternatively spliced
forms. The long form of the oxidase (lf-ALR, 23 kDa) exists predominantly in the mitochondrial
intermembrane space (IMS) and contains an 80-amino acid N-terminal extension with respect to
the short form (sf-ALR, 15 kDa) that is an extracellular cytokine and also participates in intracellu-
lar redox-dependent signaling pathways.
The crystallographic structure of sf-ALR has been determined and the coordinates deposited with
PDB code 3O55. Furthermore two mutants of sf-ALR on the cysteines of the CXXC motif have
been crystallized and their structure determined; this allowed to propose hypothesis on the molecu-
lar recognition and on the electron transfer mechanisms from MIA40 to ALR. The protein was
structurally characterized not only in the crystal state but also in solution and this has allowed to
infer hypothesis on the mechanism of molecular recognition and on electron transfer between the
partner MIA40 and ALR. The two coordinate sets have been deposited at the PDB under the fol-
lowing accession codes (3U2L, 3U2M) and the relevant papers published (1. Banci L, Bertini I,
Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Kallergi E, Lionaki E, Pozidis C, Tokatlidis K.
(2011). Proc Natl Acad Sci U S A. 108(12):4811-6 2. Banci L, Bertini I, Calderone V, Cefaro C, Cio-
fi-Baffoni S, Gallo A, Tokatlidis K. (2012). An electron-transfer path through an extended disulfide
relay system: the case of the redox protein ALR. J Am Chem Soc. 134(3):1442-5).
ALS (amyotrophic lateral sclerosis) is a neurodegenerative disease characterized by the progres-
sive dysfunction and loss of motor neurons. Insoluble protein aggregates and SOD1 oligomers
have been found in corresponding neural tissues of ALS patients. These oligomers are only found
when SOD1 is in its immature, metal-free form and the oligomerization occurs through the for-
mation of intermolecular disulfide bonds between the two free cysteines which become solvent ex-
posed in the absence of metal cofactors. Therefore, one approach to the development of strategies
for ALS treatment could be based on the prevention of the formation and on the disruption of the
intermolecular disulfide bonds in SOD1 oligomers. This can be achieved by treating with molecules
like cisplatin which bind to the cysteine sulfur, thus preventing their oxidative bridging, and which
are also capable to break disulfide bonds, therefore leading to the aggregate disassembling. The
adduct SOD1-cisPt has been crystallized and its structure determined (figure) leading to the eluci-
dation of the interaction and the binding mode of cisPt to SOD1. This has provided evidences for a
potential use of cisplatin in the treatment of ALS. The relevant paper has been published (Banci L,
Bertini I, Blaževitš O, Calderone V, Cantini F, Mao J, Trapananti A, Vieru M, Amori I, Cozzolino M,
Carrì MT. Interaction of Cisplatin with Human Superoxide Dismutase. J Am Chem Soc.) and the
coordinates deposited at the PDB under the accession code 3RE0.
Scientific Annual Report 2011
21
DRUG DISCOVERY
One of our top areas of research focuses on the design and development of new pharmaceuticals
and the identification of new therapeutic targets. Our commitment to drug discovery is evidenced
both by studies published internationally in recent years and by patents that CERM personnel have
co-authored with researchers from spin-off of the University of Florence. The strategy underpinning
the CERM-FiorGen, a non-profit pharmacogenomic foundation and several colleagues in medicine
and organic chemistry, is based on the determination of a pathology of interest and genomic analy-
sis to identify potential therapeutic targets. In silico target analysis contributes to structural model-
ing (based on the templates of homologous proteins), continuing on to molecular docking. The next
step includes on the one side cloning, expression and purification of the selected targets and on
the other side the synthesis of molecules selected through in silico rational drug design to obtain
real-time experimental data on interaction efficiencies as well as to optimize the projection of se-
cond generation molecules.
CERM is currently investigating the thermodynamic parameters that affect the ligand binding in
fragment linking approach in order to speed-up the drug discovery process. So far, CERM re-
searchers have concentrated mainly on isolated targets such as matrix metalloproteins (MMPs),
S100, phosphatases, telomerases, superoxide dismutases, RAGE receptor and HGF. Now we aim
to explore the world of extracellular protein-protein interactions. The knowledge of protein-protein
interaction networks (in particular MMPs with ECM components, S100s with RAGE receptor and
HGF with MET receptor) will increase our understanding of the molecular mechanisms underlying
specific physiopathological processes and this information will be exploited to identify new molecu-
lar targets and to design new candidate drugs. In this respect, in collaboration with University of
Cambridge, CERM researchers are carrying out the screening of libraries of chemicals to identify
inhibitors of the HGF-MET interaction. Concerning RAGE target, the whole receptor and the isolat-
ed domains have been expressed and the interaction with several pathological-related ligands al-
ready characterized.
Crystal structure of cisplatin-bound apo
hSOD1. Upper panel: ribbon representation
of one of the two dimers present in the
asymmetric unit highlighting the two Cys111
facing each other at the interface and the
cisplatin bound to them. Lower panel: insight
showing the two Cys111 bound to cisplatin
and their electron density. It is evident the
lack of the space to host two cisplatin moie-
ties at the same time. (N blue; Pt gray; Cl
green).
Scientific Annual Report 2011
22
INTERNATIONAL DOCTORATE IN MECHANISTIC AND STRUCTURAL
SYSTEMS BIOLOGY/STRUCTURAL BIOLOGY
The International Doctorate in Structural Biology is offered in collaboration with the University of
Frankfurt, Germany and Utrecht University, Netherlands. The CERM facility hosts the PhD pro-
gram, which is celebrating its 10th anniversary in 2010.
The doctorate course aims to place suc-
cessful candidates at the forefront of mod-
ern investigative methodologies in the fields
of structural biology and biotechnology.
Highlights of the curricula are:
1) Applications of nuclear magnetic
resonance spectroscopy and X-ray crystal-
lography for structural studies, function and
dynamics of biological macromolecules,
protein-protein interactions;
2) Molecular biology techniques for
preparation and manipulation of proteins,
DNA and bacterial consortia;
3) Development of new drugs through
drug design techniques, which, starting
from the structural description of proteins
and molecular mechanisms underlying the
onset and progression of diseases could
lead to the proposal of new therapeutic op-
tions;
4) Metabonomics, defined as the quanti-
tative measure of the dynamic multi-
parametric metabolic response of living
systems to pathophysiological stimuli or
genetic modification, which is emerging as a useful complement to the characterization of various
diseases
5) Bioinformatics as a strategy to study kinetic and thermodynamic phenomenon and protein
dynamics; to develop ways to use genomic databases to identify molecular targets for developing
new drugs.
The program trains outstanding students in Life Sciences and in spectroscopic methods applied to
biotechnology. The added value of the international doctorate is the development of a transnational
and multilingual training project geared at producing scholars who can assume leadership roles in
the advancement of science, research and technology across borders within the European Union
and abroad.
Full-time attendance is mandatory as is commitment to research activities. In addition to seminars
and courses, students must attend any training sessions that may be organized jointly with other
universities. PhD students should view the research seminars as a basic tool for their own training.
Every PhD student is encouraged to liaise with foreign universities and take part in teaching and
research training as well as internships abroad.
Scientific Annual Report 2011
23
EUROPEAN NMR SERVICE
The CERM Nuclear Magnetic Resonance (NMR) research infrastructure provides an ideal envi-
ronment for research in the field of NMR spectroscopy applied to metalloproteins.
The uniqueness of the infrastructure lies in the wide number of available instruments, allowing us-
ers to combine two proposed approaches to the study of paramagnetic systems, i.e. high-
resolution in solution and in solid state NMR and relaxometry technologies.
All of the high-resolution NMR
instruments are part of the digital
Bruker Avance series. Most of
them are equipped with cryo-
probes to improve the sensitivity
of 1H and/or
13C, including a pro-
totype 13
C cryoprobehead. Sev-
eral probeheads (heads measur-
ing single, double and triple
resonance, low and high fre-
quency, high power) and different
electronic configurations (number
of channels, lock-switch) are also
available to meet specific needs.
At 600 MHz spectrometer pro-
vides a card to remove radiation damping. The 500, a 600 MHz, two 700s, the 800, and the 900
MHz spectrometers are equipped with a CryoProbe. Thanks to a collaboration with Bruker, one
of the two 700 MHz spectrometers is equipped with a prototype CryoProbe for 13
C direct detention,
specifically developed for CERM. Thanks to promising results, Bruker put an optimised CryoProbe
not only for proton detection, but also for carbon (TCI) on the market; the 900 and 700 MHz spec-
trometers are equipped with this device. The highest magnetic field available for Soliution NMR is
the 950 MHz equipped with a TCI CryoProbe. A 700 MHz WB and 850 WB are dedicated to
solid-state NMR spectroscopy. The 700 MHz is equipped with a 4mm and 3.2mm MAS double and
triple resonance probes. The 850 MHz, Ultra Shielded Ultra Stabilized is equipped with 1.3 mm
and 3.2mm MAS probes for 13C, 15N and 1H detection.
With regards to low-field instruments, CERM is a unique example for the efforts made towards the
development of relaxometry. The most sensitive instrument for low-field relaxation (High Sensitiv-
ity Fast Field Cycling NMR) developed during RTD projects for the EC and patented by Stelar (It-
aly), is available, and covers the 0.01-45 MHz range.
A parallel computer, several graphic and computational stations (both Linux and Unix) and a PC
cluster provide the necessary problem-solving power to adequately complement the analysis in-
struments.
A biotechnology lab is available to users, together with other biophysical equipment for EPR, CD,
UV-vis and stopped-flow measurements. Users can also access other university infrastructures,
such as those of mass spectrometry, x-ray diffractometry, Raman resonance, and non-linear spec-
troscopies.
Access to European users is funded by the European Commission since 1994. In 2011 this funding
was provided through two different programs: EastNMR (www.east-nmr.eu) and BioNMR
(www.bio-nmr.net). Overall, under the two programs, 326 days of machine time were granted to
European users for 19 different research projects.
Scientific Annual Report 2011
24
NMR NATIONAL SERVICE
In 2011, several national users from public and private laboratories used instruments that CERM
placed at their disposal.
Although the MIUR – FIRB Project, that provided Access to the Users, has been completed in June
2010, this laboratory decided to maintain its instrumentation available to all National Users, pro-
vided their research project matched quality criteria in terms of scientific interest, excellence and
feasibility.
The NMR National Service protocol provides access to all instruments and establishes that the
days of machine-time assigned to users must include Technical Assistance for both spectra acqui-
sition and elaboration. Users can use CERM’s chemistry and biochemistry labs to complete their
sample preparation or carry out necessary chemical or biochemical procedures. National Service
users can access the instruments and labs of several Interdepartmental Centres in strict collabora-
tion with CERM, such as the Genomics and Proteomics Centre GeneExpress, the Interdepartmen-
tal Centre of Mass Spectrometry, and all instruments available at the University of Florence’s De-
partment of Chemistry. All users who used the NMR National Service requested access through an
on-line presentation of a research proposal with an application for machine-time that was complete
from both a qualitative point of view (requested spectrometer and type of experiments) and a quan-
titative point of view (number of days requested). Applications were considered and accepted by a
local panel, composed of members of the CERM Board of Directors and another panel made up of
national experts. Both panels gave favorable opinions, respectively, based on technical feasibility
of the scientific relevance and the appropriateness of the application with the presented project.
Overall, National academic visitors access to the Facility for a total of 43 days. The Access from
Users from private entities has been substantially maintained with respect to 2010.
The overall days of machine time that CERM made available to national members, whether from
public research laboratories or private entities (without considering CIRMMP and CERM Associ-
ates such as scientists from Bologna or from Fiorgen), amount to 161.
Scientific Annual Report 2011
25
COLLABORATIVE RESEARCH CERM has several collaborations, partnerships and joint projects with national and international
industries with the role of developing new processes, new methodologies and new approaches
employed by industrial partners. In particular:
NOVARTIS VACCINES
In conjunction with Novartis-Vaccines, CERM is carrying out a research project that aims to use an
integrated approach in order to select antigens that are promising vaccine candidates against in-
fectious bacterial diseases. In particular, the project aims at developing a vaccine against two in-
fectious agents, Nesseria menengitidis of the Group B serotype and Clostridium difficile. Both are
responsible for important human diseases for which no effective preventive therapy is available.
CERM researchers have solved the 3D structure of fhbp (Factor H binding protein) from N.
Meningitidis using NMR spectroscopy. fHbp is expressed by all the pathogenic strains of N.
Meningitidis and can be classified in three distinct sequence variants. fHbp is able to induce anti-
bodies that mediate complement bacteriloysis and, at the same time, it is also able to bind human
factor H, a negative regulator of the alternative complement pathway. These properties make fHpb
one of the most promising antigens as a component for a universal vaccine against
Meningococcus B. The fHbp structural characterization performed at CERM, allowed for the identi-
fication of a conformational protective epitope on the protein surface and to undertake a follow-up
study, currently in progress, in order to develop a second-generation vaccine with higher stability
and greater range of coverage. The research performed at CERM, in collaboration with Novartis
Vaccines, increased our knowledge regarding the features of the antigenic protein fHbp, allowing
the design of molecules able to elicit cross-protective bactericidal
Scientific Annual Report 2011
26
FIORGEN
The Pharmacogenomics FiorGen Onlus Foundation (www.fiorgen.net) is a non-profit foundation
that promotes studies in predictive and personalized medicine. The Foundation conducts research
in the application of genomic and molecular data to help find new products, and to help determine
the susceptibility to a particular disease or disorder. CERM FiorGen and work together on the main
lines of structural biology and metabolomics.
The study of diseases through the holistic approach of metabolomics can be very important to ob-
tain new information on their mechanisms and their impact on human health.
The FiorGen foundation conducted during 2011 metabolomic studies on breast cancer, investigat-
ing the correlation between metabolic profile and the probability of postoperative recurrence, and
on colorectal cancer, saying that the metabolic profile is a good predictor for the time survival of
metastatic patients.
During the 2011 has been published several paper in the field of metabolomics: one paper on the
metabolic fingerprint of breast cancer patients, one paper on the potential celiac subjects, one
study on the metabolic profile of the cardiovascular risk and one paper, performed on saliva sam-
ples, about the metabolic profile of chronic periodontitis.
All the papers published with the contribution of the FiorGen Foundation are significant for the de-
velopment of the respective scientific fields and are published on high impact factor journals.
CERM houses the data processing center and the main biorepository of the da Vinci European
BioBank (daVEB; https://www.davincieuropeanbiobank.org), owned by FiorGen. The interaction
between daVEB and CERM is strategic and synergistic. Scientific collaborations in the
metabolomic filed contribute to the development of SOPs validated by NMR and to the enrichment
in terms of type and number of samples stored in the biobank, which currently houses a collection
of unique samples (biofluids, tissue and DNA) of growing importance by number in the following
areas: melanoma, rare skin diseases, diseases of the genital-urinary cancer, diseases of the car-
dio-circulatory, digestive diseases, breast cancer, non-Hodgkin's lymphoma, diseases associated
with aging bone. On the other hand, the biobank acts as a support to the metabolomics research
via NMR carried out at CERM by providing a conservation service of samples and associated data
following protocols in accordance with international standards.
Scientific Annual Report 2011
27
PROTERA ??????????????????
ProtEra (http://www.proterasrl.com) is a biotech company which focuses on the study of new drug
targets and the development of novel methods for defining new drug candidates. The core busi-
ness of Protera is drug discovery aimed at finding selective and effective molecules with high ther-
apeutic potential. The pipeline includes target identification, structure identification, in-silico screen-
ing and in-vivo structure determination, characterization of target ligand-adducts, organic synthesis
and preclinical development.
In 2010, research projects at ProtEra include:
- Anticancer Prodrug: a cytotoxic compound released from the telomerase catalytic activity which
can be used to treat solid tumors, blood and blood derivatives in patients with retro viral infections
(HIV). 2010 marked significant advancements in research on tumors such as pancreatic adenocar-
cinoma, which is normally resistant to chemotherapy.
- Antiaging, Caries, and Periodontitis: the company has developed metal proteinase inhibitors that
could have important applications in both pharmaceutical and cosmetic treatment of the diseases.
Preclinical molecular development has been promising (for solubility, bioavailability, activity and
easy synthesis) and trials have begun in rodents for acute toxicity (oral, intravenous, intradermic
and toxicocinetic) in GLP.
ProtEra took part in the following European project together with CERM:
- EPISODE: a project coordinated by the Tuscan Regional Government aimed at fostering interre-
gional business and technology transfer between the regions of Tuscany, Campania and Berlin-
Brandenburg.
- IDPbyNMR: is a project that is part of the Marie Curie Action for Initial Training Network (ITN) in
the 7th Framework Programme of the European Commission http://www.idpbynmr.eu/
Scientific Annual Report 2011
28
PROTEIN STRUCTURES 2011
Banci, L., Bertini, I., Ciofi-Baffoni, S.,
Kozyreva, T., Mori, M., and Wang, S.
SCO proteins are involved in electron
transfer processes
J.Biol.Inorg.Chem., 13: 391-403, 2011
PDB code: 2L4D
Gentile, M. A., Melchiorre, S., Emolo, C.,
Moschioni, M., Gianfaldoni, C., Pancotto,
L., Ferlenghi, I., Scarselli, M., Pansegrau,
W., Veggi, D., Merola, M., Cantini, F., Rug-
giero, P., Banci, L., and Masignani, V.
Structural and functional
characterization of the Streptococcus
pneumoniae RrgB pilus backbone D1
domain,
J.Biol.Chem., 286: 14588-14597, 2011
PDB code: 2L4O
Banci L, Bertini I, Calderone V, Cefaro C,
Ciofi-Baffoni S, Gallo A, Kallergi E, Lionaki
E, Pozidis C, Tokatlidis K.
Molecular recognition and substrate
mimicry drive the electron-transfer pro-
cess between MIA40 and ALR
Proc.Natl.Acad.Sci. USA., 108: 4811-4816,
2011 PDB code: 3O55
Benini, S., Cianci, M., Ciurli, S.
Holo-Ni(2+)Helicobacter pylori NikR
contains four square-planar nickel-
binding sites at physiological pH
Dalton Trans., 40: 7831-7833, 2011
PDB code: 2Y3Y
Scientific Annual Report 2011
29
Banci, L., Bertini, I, Ciofi-Baffoni, S.,
Boscaro, F., Chatzi, A., Mikolajczyk, M.,
Tokatlidis, K., Winkelmann, J.
Anamorsin is a [2Fe-2S] cluster-
containing substrate of the Mia40-
dependent mitochondrial protein
trapping machinery
Chem. Biol., 18: 794-804, 2011
PDB code: 2LD4
Scarselli, M., Arico, B., Brunelli, B., Savino,
S., Di Marcello, F., Palumbo, E., Veggi, D.,
Ciucchi, L., Cartocci, E., Comanducci, M.,
Giuliani, M. M., Cantini, F., Dragonetti, S.,
Colaprico, A., Doro, F., Giannetti, P.,
Pallaoro, M., Brogioni, B., Tontini, M.,
Hilleringmann, M., Nardi Dei, V., Banci, L.,
Pizza, M. G., and Rappuoli, R.
Structure-based design of a
meningococcal antigen inducing broad
protective immunity
Sci.Trans.Medicine, 3, 91ra62, 2011.
PDB code: 2Y7S
Docquier, J. D., Benvenuti, M., Calderone,
V., Rossolini, G. M., and Mangani, S.
Structure of the extended-spectrum L-
lactamase TEM-72 inhibited by citrate
Acta Crystallograph. Sect.F.
Struct.Biol.Cryst.Commun., 67: 303-306,
2011
PDB code: 3P98
De Luca, F., Benvenuti, M., Carboni, F.,
Pozzi, C., Rossolini, G. M., Mangani, S.,
and Docquier, J. D.
Evolution to carbapenem-hydrolyzing in
noncarbapenemase class D beta-
lactamase by rational protein design
Proc.Natl.Acad.Sci.USA, 108: 18424-
18429, 2011 PDB code: 3QNC
Aachmann, F. L., Del Conte, R., Kim, H-Y.,
Gladyshev, V. N., Dikiy, A.
Structural and biochemical analysis of
mammalian methionine sulfoxide
reductase B2
Proteins, 79: 3123-3131, 2011
Scientific Annual Report 2011
30
PDB code: 2L1U Banaszak, K., Martin-Diaconescu. V.,
Bellucci. M., Zambelli, B., Rypniewski, W.,
Maroney, M. J., Ciurli, S.,
Crystallographic and X-ray absorption
spectroscopic characterization of
Helicobacter pylori UreE bound to Ni2+
and Zn2+ reveal a role for the disordered
C-terminal arm in metal trafficking
Biochem. J., 441: 1017-1026, 2012 PDB code: 3TJ8, 3TJ9, 3TJA
Tottey, S., Patterson, A. M., Banci, L.,
Bertini, I., Felli, I. C., Pavelkova, A., Dainty,
S. J., Pernil, R., Waldron, K. J., Foster, A.
W., Robinson, N. J.
Cyanobacterial metallochaperone
inhibits deleterious side reactions of
copper
Proc.Natl.Acad.Sci.USA, 109: 95-100,
2012
PDB code: 2LDI
Bertini, I., Calderone, V., Cerofolini, L.,
Fragai, M., Geraldes, C. F. G. C., Hermann,
P., Luchinat, C., Parigi, G., and Teixeira, J.
M. C.
The catalytic domain of MMP-1 studied
through tagged lanthanides
FEBS Lett., 586: 557-567, 2012
PDB code: 3SHI
Banci, L., Bertini, I., Cefaro, C., Ciofi-
Baffoni, S., Gallo, A.
The functional role of two interhelical
disulfide bonds in human Cox17 from a
structural perspective
J.Biol.Chem., 286: 34382-34390, 2011
PDB code: 2LGQ
Scientific Annual Report 2011
31
Docquier, J.D., Benvenuti, M., Calderone,
V., Stoczko, M., Menciassi, N., Rossolini,
G.M., Mangani, S.,
High-resolution crystal structure of the
subclass B3 metallo-beta-lactamase
BJP-1: rational basis for substrate speci-
ficity and interaction with sulfonamides.
Antimicrob.Agents Chemother.54: 4343-
4351, 2010
PDB code: 3M8T, 3LVZ
Scientific Annual Report 2011
32
MEETINGS
Archiving and exchanging (NMR) metabolomics data, Magnetic Resonance Center(CERM),
University of Florence, Sesto FIorentino (Florence), Italy, November 18, 2011
WeNMR Workshop - Computational aspects of the joint use of SAXS and NMR (May 27th 2011)
SEMINARS
November 30th Prof. Gregg B. Fields (Torrey Pines Institute for Molecular Studies Port St. Lucie,
Florida, USA) - “Exosite Inhibitors of Metalloproteinases"
November 25th Prof. Harald Schwalbe (University of Frakfurt, Germany) - “Investigation of disulfide
bond formation on unfolded state structure and dynamics"
November 16th Prof. Joan S. Valentine (UCLA - Department of Chemistry and Biochemistry, USA)
- “Metal ions and misfolding in SOD-linked ALS"
November 14th Prof. Astrid Gräslund (Stockholm University - Dept. of Biochemistry and
Biophysics, Sweden) - “NMR studies of the amyloid beta peptide involved in Alzheimer´s
disease:Molecular interactions, secondary structure conversions and aggregation"
November 4th Dr. Jochem Struppe (Bruker Biospin Corp., USA) - “Practical aspects in Biological
Solid State NMR"
October 24th Prof. Julia S. Johansen (University of Copenhagen, Denmark) - “YKL-40a protein
associated with death and life"
July 14th Prof. Robert G. Griffin (Massachusetts Institute of Technology, USA) - “SACCONI
MEDAL LECTURER 2011"
June 20th Prof. Janez Plavec (head of NMR centre, National Institute of Chemistry Ljubljana,
Slovenia) - “Cation localization and movements within G-quadruplexes"
June 7th Dr. Sonia Longhi (AFMB, UMR 6098, CNRS et Aix-Marseille Université) - “Structural
disorder within the replicative complex of paramyxoviruses"
May 17th Prof. K. V. R. Chary (Tata Institute of Fundamental Research, Mumbai, India) -
“TWENTY-20 NMR: Rapid Data acquisition methods in Protein NMR"
May 12th Prof. Dr. Bernd Schultes (Head of the Interdisciplinary Obesity Center Kantonsspital St.
Gallen - Rorschach, Switzerland) - “Pathophysiology of obesity and effects of surgically-induced
weight loss"
April 29th Prof. Patrizia Brigidi (Department of Pharmaceutical Sciences, University of Bologna)
“Studying the human intestinal microbiota in the meta-omics era"
April 20th Prof. Toshiya Endo (Department of Chemistry - Nagoya University, Japan)“Structural
biology of mitochondrial protein import”
Scientific Annual Report 2011
33
April 18th Prof. Gianni Fochi, Scuola Normale Superiore di Pisa, “Fischi per fiaschi: quando il
linguaggio comune provoca lo scienziato ”
April 13th Prof. Carla Koehler (Department of Chemistry and Biochemistry - UCLA (USA) "A
chemical biology approach for studying mitochondrial redox regulation"
April 4th Prof. Nigel Robinson, Department of Chemistry, Durham University (UK), "How cells help
proteins toacquire the correct metals" THE LUIGI SACCONI MEMORIAL LECTURE IN
CHEMISTRY
March 18th Prof. Lyndon Emsley (Ecole Normale Supérieure de Lyon) "Protein NMR
Crystallography"
March 7th Prof. Roland Lill (Institut für Zytobiologie und Zytopathologie, Universität Marburg)
"Biogenesis of iron-sulfur proteins in eukaryotes: components, mechanisms and associated
diseases"
March 2nd Prof. Antonio Randazzo (Dipartimento di Chimica delle Sostanze Naturali, Università
degli Studi di Napoli) & Dr. Luciana Marinelli (Dipartimento di Chimica Farmaceutica e
Tossicologica, Università degli Studi di Napoli) "G-quadruplex DNA for drug discovery and
applications"
February 11th Prof. Dr. Michele Parrinello (ETH Zurich) "Proteins in motion"
February 2nd Prof. Sergio Carrà (Politecnico di Milano, Italy) - "Integrazione culturale e sviluppo
economico: ruolo delle scienze chimiche dall’unificazione a oggi"
January 31st Prof. Maurizio Vecchi (University of Milan and IRCCS Policlinico San Donato di
Milano) "Chronic Inflammatory Bowel Diseases and Gut Microflora"
January 18th Prof. Gianni Fochi (Scuola Normale Superiore di Pisa) "Fischi per fiaschi: quando il
linguaggio comune provoca lo scienziato"
January 13th Prof. Gianfranco Scorrano (University of Padua, Italy) - "Firenze, la Gazzetta Chimi-
ca e l’Unificazione dell’Italia"
Scientific Annual Report 2011
34
Publ ications 2011 (Ordered by decreasing impact factor)
1. Scarselli, M., Arico, B., Brunelli, B., Savino, S., Di Marcello, F., Palumbo, E., Veggi, D.,
Ciucchi, L., Cartocci, E., Bottomley, M. J., Malito, E., Lo, Surdo P., Comanducci, M., Giuliani,
M. M., Cantini, F., Dragonetti, S., Colaprico, A., Doro, F., Giannetti, P., Pallaoro, M., Brogioni,
B., Tontini, M., Hilleringmann, M., Nardi-Dei, V., Banci, L., Pizza, M., and Rappuoli, R., Ratio-
nal design of a meningococcal antigen inducing broad protective immunity, Sci.Transl.Med., 3,
91ra62-, 2011
2. Zambelli, B., Musiani, F., Benini, S., and Ciurli, S., Chemistry of Ni2+ in urease: sensing,
trafficking and catalysis, Acc.Chem.Res., 44, 520-530, 2011 (IF 18.203).
3. Bertini, I., Felli, I. C., Gonnelli, L., Vasantha Kumar, M. V., and Pierattelli, R., 13C direct-
detection biomolecular NMR spectroscopy in living cells, Angew.Chem.Int.Ed., 50, 1-4, 2011
(IF 12.703).
4. Knight, M. J., Webber, A. L., Pell, A. J., Guerry, P., Barbet-Massin, E., Bertini, I., Felli, I. C.,
Gonnelli, L., Pierattelli, R., Emsley, L., Lesage, A., Hermann, T., and Pintacuda, G., Fast reso-
nance assignment and fold determination of human superoxide dismutase by high-resolution
proton-detected solid state MAS NMR spectroscopy, Angew.Chem.Int.Ed., 50, 11697-11701,
2011 (IF 12.703).
5. Polvani, S., Calamante, M., Foresta, V., Ceni, E., Mordini, A., Quattrone, A., D'Amico, M.,
Luchinat, C., Bertini, I., and Galli, A., Acycloguanosyl 5 -thymidyltriphosphate, a new thymidine
analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice, Gastro-
enterology, 140, 709-720, 2011 (IF 12.403).
6. Bertini, I., Luchinat, C., and Parigi, G., Moving the frontiers in solution solid state bioNMR.
A celebration of Harry Gray's 75th birthday, Coord.Chem.Rev., 255, 649-663, 2011 (IF
10.018).
7. Banci, L., Bertini, I., Calderone, V., Cefaro, C., Ciofi-Baffoni, S., Gallo, A., Kallergi, E.,
Lionaki, E., Pozidis, C., and Tokatlidis, K., Molecular recognition and substrate mimicry drive
the electron-transfer process between MIA40 and ALR, Proc.Natl.Acad.Sci.USA, 108, 4811-
4816, 2011 (IF 9.771).
8. Bertini, I., Luchinat, C., Parigi, G., Ravera, E., Reif, B., and Turano, P., Solid-state NMR of
proteins sedimented by ultracentrifugation, Proc.Natl.Acad.Sci.USA, 108, 10396-10399, 2011
(IF 9.771).
9. Cardinale, D., Guaitoli, G., Tondi, D., Luciani, R., Henrich, S., Salo-Ahen, O. M., Ferrari, S.,
Marverti, G., Guerrieri, D., Ligabue, A., Frassineti, C., Pozzi, C., Mangani, S., Fessas, D.,
Guerrini, R., Ponterini, G., Wade, R. C., and Costi, M. P., Protein-protein interface-binding pep-
tides inhibit the cancer therapy target human thymidylate synthase, Proc.Natl.Acad.Sci.USA,
108, E542-549, 2011 (IF 9.771).
Scientific Annual Report 2011
35
10. De Luca, F., Benvenuti, M., Carboni, F., Pozzi, C., Rossolini, G. M., Mangani, S., and
Docquier, J. D., Evolution to carbapenem-hydrolyzing in non-carbapenemase class D beta-
lactamase by rational protein design and loop grafting, Proc.Natl.Acad.Sci.USA, 108, 18424-
18429, 2011 (IF 9.771).
11. Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Losacco, M., and Natile, G., Probing the in-
teraction of cisplatin with the human copper chaperone Atox1 by solution and in-cell NMR
spectroscopy, J.Am.Chem.Soc., 133, 18361-18369, 2011 (IF 9.019).
12. Banci, L., Bertini, I., Blazevits, O., Cantini, F., Lelli, M., Luchinat, C., Mao, J., and Vieru, M.,
NMR characterization of a "fibril-ready" state of demetallated wild-type superoxide dismutase,
J.Am.Chem.Soc., 133, 345-349, 2011 (IF 9.019).
13. Bertini, I., Gonnelli, L., Luchinat, C., Mao, J., and Nesi, A., A new structural model Aß40
fibrils, J.Am.Chem.Soc., 133, 16013-16022, 2011 (IF 9.019).
14. Corzilius, B., Smith, A. A., Barnes, A. B., Luchinat, C., Bertini, I., and Griffin, R. G., High-
filed dynamic nuclear polarization with high spin transition metal ions, J.Am.Chem.Soc., 133,
5648-5651, 2011 (IF 9.019).
15. Mastarone, D. J., Harrison, V. S. R., Eckermann, A. L., Parigi, G., Luchinat, C., and
Meade, T. J., A modular system for the synthesis of multiplexed MR probes, J.Am.Chem.Soc.,
133, 5329-5337, 2011 (IF 9.019).
16. Oakman, C., Tenori, L., Claudino, W. M., Cappadona, S., Nepi, S., Battaglia, A., Bernini,
P., Zafarana, E., Saccenti, E., Fornier, M., Morris, P. G., Biganzoli, L., Luchinat, C., Bertini, I.,
and Di Leo, A., Identification of a serum-detectable metabolomic fingerprint potentially correlat-
ed with the presence of micrometastatic disease in early breast cancer patients at varying risks
of disease relapse by traditional prognostic methods, Ann.Oncol., 22, 1295-1301, 2011 (IF
6.452).
17. Jones, J. E., Amoroso, A. J., Dorin, I. M., Parigi, G., Ward, B. D., Buurma, N. J., and Pope,
S. J. A., Bimodal, dimetallic lanthanide complexes that bind to DNA: the nature of binding and
its influence on water relaxivity, Chem.Commun., 47, 3374-3376, 2011 (IF 5.787).
18. Banci, L., Bertini, I., Ciofi-Baffoni, S., Boscaro, F., Chatzi, A., Mikolajczyk, M., Tokatlidis,
K., and Winkelmann, J., Anamorsin is a 2Fe2S cluster-containing substrate of the Mia40-
dependent mitochondrial protein trapping machinery, Chem.Biol., 18, 794-804, 2011 (IF
5.603).
19. Bernini, P., Bertini, I., Calabro, A., La Marca, G., Lami, G., Luchinat, C., Renzi, D., and
Tenori, L., Are patients with potential celiac disease really potential? The answer of
metabonomics, J.Proteome Res., 10, 714-721, 2011 (IF 5.460).
20. Musiani, F., Ciurli, S., and Dikiy, A., The Interaction of selenoprotein W with 14-3-3 pro-
teins: a computational approach, J.Proteome Res., 10, 968-976, 2011 (IF 5.460).
21. Bernini,P.; Bertini,I.;Luchinat,C.;Tenori,L.;Tognaccini,A.; The cardiovascular risk of healthy
individuals studied by NMR metabonomics of plasma samples, J.Proteome Res., 10, 4983-
4992, 2011 (IF 5.460).
Scientific Annual Report 2011
36
22. Ardà, A., Canada, F. J., Nativi, C., Francesconi, O., Gabrielli, G., Ienco, A., Jimenez-
Barbero, J., and Roelens, S., Chiral diaminopyrrolic receptors for selective recognition of
mannosides. Part 2: a view of the recognition modes by X-ray, NMR; spectroscopy and molec-
ular modeling, Chem.Eur.J., 17, 4821-4829, 2011 (IF 5.45).
23. Nativi, C., Francesconi, O., Gabrielli, G., Vacca, A., and Roelens, S., Chiral
diamonopyrrolic receptors for selective recognition of mannosides. Part 1: design, synthesis
and affinities of second-generation tripodal receptors, Chem.Eur.J., 17, 4814-4820, 2011 (IF
5.45).
24. Gentile, M. A., Melchiorre, S., Emolo, C., Moschioni, M., Gianfaldoni, C., Pancotto, L.,
Ferlenghi, I., Scarselli, M., Pansegrau, W., Veggi, D., Merola, M., Cantini, F., Ruggiero, P.,
Banci, L., and Masignani, V., Structural and functional characterization of the Streptococcus
pneumoniae RrgB pilus backbone D1 domain, J.Biol.Chem., 286, 14588-14597, 2011 (IF
5.328).
25. Banci, L., Bertini, I., Cefaro, C., Ciofi-Baffoni, S., and Gallo, A., Functional role of two
interhelical disulfide bonds in human Cox17 protein from a structural perspective,
J.Biol.Chem., 286, 34382-34390, 2011 (IF 5.328).
26. Mangani, S., Cancian, L., Leone, R., Pozzi, C., Lazzari, S., Luciani, R., Ferrari, S., and
Costi, M. P., Identification of the binding modes of N-phenylphthalimides inhibiting bacterial
thymydilate synthase through X-ray crystallography screening, J.Med.Chem., 54, 5454-5467,
2011 (IF 5.207).
27. Bertini, I., Case, D. A., Ferella, L., Giachetti, A., and Rosato, A., A grid-enable web portal
for NMR structure refinement with AMBER, Bioinformatics, 27, 2384-2390, 2011 (IF 4.877).
28. Andreini, C., Bertini, I., and Cavallaro, G., Minimal functional sites allow a classification of
zinc sites in proteins, Plos ONE, 10, e26325-, 2011 (IF 4.411).
29. Banci, L., Barbieri, L., Bertini, I., Cantini, F., and Luchinat, E., In-cell NMR in E.coli to moni-
tor maturation steps of hSOD1, Plos ONE, 6, e23561-, 2011 (IF 4.411).
30. Bertini, I., Chevance, S., Del Conte, R., Lalli, D., and Turano, P., The anti-apoptotic Bcl-xL
protein, a new piece in the puzzle of cytochrome c interactome, Plos ONE, 6, e18329-, 2011
(IF 4.411).
31. Wegerich, F., Turano, P., Allegrozzi, M., Möhwald, H., and Lisdat, F., Electroactive multi-
layer assemblies of bilirubin oxidase and human cytochrome c mutants: insight in formation
and kinetic behavior, Langmuir, 27, 4202-4211, 2011 (IF 4.268).
32. Benini, S., Cianci, M., and Ciurli, S., Holo-Ni2+ helicobacter pylori NikR contains four
square-planar nickel-binding sites at physiological pH, Dalton Trans., 40, 7831-7833, 2011 (IF
4.081).
33. Manet, I., Manoli, F., Zambelli, B., Andreano, G., Masi, A., Cellai, L., and Monti, S., Affinity
of the anthracycline drugs doxorubicin and sabarubicin for human telomeric G-quadruplex
structures, Phys.Chem.Chem.Phys., 13, 540-551, 2011 (IF). 4.064
Scientific Annual Report 2011
37
34. Bertini, I., Felli, I. C., Gonnelli, L., Vasantha Kumar, M. V., and Pierattelli, R., High-
resolution characterization of intrinsic disorder in proteins, ChemBioChem,
DOI:10.1002./cbic.201100406, 2011 (IF 3.945).
35. Banci, L., Bertini, I., Ciofi-Baffoni, S., D'Alessandro, A., Jaiswal, D., Marzano, V., Neri, S.,
Ronci, M., and Urbani, A., Copper exposure effects on yeast mitochondrial proteome,
J.Proteomics , 74, 2522-2535, 2011 (IF 3.851).
36. Andreini, C., Bertini, I., Cavallaro, G., Decaria, L., and Rosato, A., A simple protocol for the
comparative analysis of the structure and occurence of biochemical pathways across
superkingdoms, J.Chem.Inf.Model., 51, 730-738, 2011 (IF 3.822).
37. Musiani, F., Bellucci, M., and Ciurli, S., Model structures of Helicobacter pylori UreD(H)
domains: a putative molecular recognition platform, J.Chem.Inf.Model., 51, 1513-1520, 2011
(IF 3.822).
38. Aimetti, M., Cacciatore, S., Graziano, A., and Tenori, L., Metabonomic analysis of saliva
reveals generalized chronic periodontitis signature, Metabolomics, DOI: 10.1007/s11306-
011.0331-2, 2011 (IF 3.608).
39. Bertini, I., Cavallaro, G., and Rosato, A., Principles and patterns in the interaction between
mono-heme cytochrome c and its partners in electron transfer processes, Metallomics, 3, 354-
362, 2011 (IF 3.592).
40. Decaria, L., Bertini, I., and Williams, R. J., Copper proteomes, phylogenetics and evolution,
Metallomics, 3, 56-60, 2011 (IF 3.592).
41. Cacciarini, M., Nativi, C., Norcini, M., Staderini, S., Francesconi, O., and Roelens, S.,
Pyrrolic tripodal receptors for carbohydrates. Role of functional groups and binding geometry
on carbohydrate recognition, Org.Biomol.Chem., 9, 2085-, 2011 (IF 3.451).
42. Babini, E., Bertini, I., Borsi, V., Calderone, V., Hu, X., Luchinat, C., and Parigi, G., Structur-
al characterization of human S100A16, a low affinity calcium binder, J.Biol.Inorg.Chem., 16,
243-256, 2011 (IF 3.287).
43. Banci, L., Bertini, I., Ciofi-Baffoni, S., Kozyreva, T., Mori, M., and Wang, S., SCO proteins
are involved in electron transfer processes, J.Biol.Inorg.Chem., 13, 391-403, 2011 (IF 3.287).
44. Lee, D.-W., El Khoury, Y., Francia, F., Zambelli, B., Ciurli, S., Venturoli, G., Hellwig, P., and
Daldal, F., Zinc inhibition of bacterial cytochrome bc1 reveals the role of E295 in proton release
at the Qo site, Biochemistry, 50, 4263-4272, 2011 (IF 3.226).
45. Bertini, I., Banci, L., Cavallaro, G., and Ciofi-Baffoni, S., Seeking the determinants of the
elusive functions of Sco proteins, FEBS J., 278, 2244-2262, 2011 (IF 3.129).
46. Bernini, P., Bertini, I., Luchinat, C., Nincheri, P., Staderini, S., and Turano, P., Standard
operating procedures for pre-analytical handling of blood and urine for metabolomic studies
and biobanks, J.Biomol.NMR, 49, 231-243, 2011 (IF 3.047).
47. Das Gupta, S., Hu, X., Keizers, P. H. J., Liu, W.-M., Luchinat, C., Nagulapalli, M., Over-
hand, M., Parigi, G., Sgheri, L., and Ubbink, M., Narrowing the conformational space sampled
by two-domain proteins with paramagnetic probes in both domains, J.Biomol.NMR, 51, 253-
263, 2011 (IF 3.047).
Scientific Annual Report 2011
38
48. Gladyshev, V. N., Kim, H-Y., Aachmann, F. L., Del Conte, R., and Dikiy, A., Structural and
biochemical analysis of mammalian methionine sulfoxide reductase B2, Proteins: Structure,
Function, and Bioinformatics, 79, 3123-3131, 2011 (IF 2.813)
49. Manet, I., Manoli, F., Zambelli, B., Andreano, G., Masi, A., Cellai, L., Ottani, S., Marconi,
G., and Monti, S., Complexes of the antitumoral drugs doxorubicin and sabarubicin with
telomeric G-quadruplex in basket conformation: ground and excited state properties,
Photochem.Photobiol.Sci., 10, 1236-1237, 2011 (IF 2.378).
50. Marconi, G., Mezzina, E., Manet, I., Manoli, F., Zambelli, B., and Monti, S., Stereoselective
interaction of ketoprofen enantiomers with a-cyclodextrin: ground state binding and photo-
chemistry, Photochem.Photobiol.Sci., 10, 48-59, 2011 (IF 2.287).
51. Richichi, B., Luzzatto, L., Notaro, R., La Marca, G., and Nativi, C., Synthesis of the essen-
tial core of the human glycosylphosphatidylinositol (GPI) anchor, Bioorg.Chem., 39, 88-93,
2011 (IF 1.59).
52. Babini, E., Hu, X., Parigi, G., and Vignali, M., Human multiprotein bridging factor 1 and
calmodulin do not interact in vitro as confirmed by NMR spectroscopy and CaM-agarose affini-
ty chromatography, Protein Expr.Purif., 80, 1-7, 2011 (IF 1.644).
53. Docquier, J. D., Benvenuti, M., Calderone, V., Rossolini, G. M., and Mangani, S., Structure
of the extended-spectrum L-lactamase TEM-72 inhibited by citrate, Acta
Crystallograph.Sect.F.Struct.Biol.Cryst.Commun., 67, 303-306, 2011 (IF 0.563).
54. Bertini, I., Emsley, L., Felli, I. C., Laage, S., Lesage, A., Lewandoski, D. A., Marchetti, A.,
Pierattelli, R., and Pintacuda, G., High-Resolution and sensitivity through-bond correlations in
ultra-fast MAS solid-state NMR, Chem.Sci., 2, 345-348, 2011