Scientific Annual Report 2011 · Scientific Annual Report 2011 2 Acknowledgements ... Prof. Antonio...

Scientific Annual Report

2011

C.E.R.M.

Centro Risonanze Magnetiche

Scientific Annual Report 2011

1

Financers

European Commission


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


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.


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.

http://www.cerm.unifi.it/


5


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


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. 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


Siena

Prof. Gianni Valensin

Utrecht

Prof. Rolf Boelens

Frankfurt

Prof. Harald Schwalbe

PANEL FOR NATIONAL ACCESS Prof. Henriette Molinari - Verona

Prof. Lucia Zetta - Milan


8

Staff


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. 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


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


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


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.


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.


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).


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.


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.


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.


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.


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


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.


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).


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.


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.


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.


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


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.


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/


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

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Banci%20L%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bertini%20I%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Calderone%20V%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Cefaro%20C%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ciofi-Baffoni%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gallo%20A%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kallergi%20E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lionaki%20E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lionaki%20E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Pozidis%20C%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Tokatlidis%20K%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Benini%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Cianci%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ciurli%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed/21725560


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

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Banci%20L%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bertini%20I%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ciofi-Baffoni%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Tokatlidis%20K%22%5BAuthor%5D


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

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Banaszak%20K%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Martin-Diaconescu%20V%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bellucci%20M%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Zambelli%20B%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Rypniewski%20W%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Maroney%20MJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ciurli%20S%22%5BAuthor%5D


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


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”


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"


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).


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).


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


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).


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

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