2nd

Annual European MicroCal Meeting

Paris, September 26-27, 2016

Welcome to the 2nd Annual European MicroCal Meeting

On behalf of Malvern Instruments, Institut Pasteur, the ARBRE-MOBIEU COST Action and the organizing committee, it is our pleasure to welcome you to MicroCal European Users’Meeting 2016 in Paris.

We would like to thank all our attendees and speakers. The organization of this meeting would not have been possible without their support.

We do hope that you will enjoy intense and stimulating discussions as well as an unforgettable stay in the light city Paris...

Introduction

The second Annual European MicroCal Meeting, co-organized by Malvern Instruments, Institut Pasteur and ARBRE European molecular biophysics network, will focus on applications, best practices, advanced data analysis and latest developments in isothermal titration and differential scanning calorimetry (ITC and DSC) and will bring together MicroCal users from across Europe.

The 2-days event will include ITC and DSC workshops and scientific sessions with panel discussions focused on the data quality, best experimental practices, new applications and advanced data analysis.

It allows users to share and broaden their experiences and network on these technologies during application-specific oral presentations, poster sessions, panel discussions and workshops. Attend seminars and roundtables with European microcalorimetry experts. Meet MicroCal application scientists and discover several microcalorimetric systems on display.

Location

Institut Pasteur Paris, 28 rue du Docteur Roux, 75015 Paris http://www.pasteur.fr/en/adress-and-access

Venue

By Metro or Bus: By Metro: Get off at either Volontaires (line 12) or Pasteur (lines 6 and 12) station. These stations are just a 5 minute walk from the Institut Pasteur. Map of the metro network: http://www.ratp.fr/en/ratp/c_5000/accueil By Bus: Bus number 95 (Institut Pasteur stop, direction Porte de Vanves) stops in front of the Institute’s entrance. Bus numbers 39 and 70 (Volontaires-Vaugirard or Sèvres-Lecourbe stops) stop slightly further away (5 to 10 minute walk) and not far from Pasteur metro station. Map of bus routes: http://www.ratp.fr/en/ratp/c_5000/accueil Note: One ticket allows you to travel on the metro and includes all connections as long as you do not leave the metro network. For most bus and tram routes in Paris and Ile-de-France, one ticket is valid for a single journey without connections regardless of length. On some routes (Balabus, Noctambus, Orlybus, Roissybus, 221, 297, 299, 350 and 351) fares depend on journey length. By car: Rue du Docteur Roux is a one way street and you pay for parking spaces. Paying public car parks are available at the following addresses: Sogeparc France 81 r Falguière - 75015 Paris Tel.: +33 (0)1 43 35 27 69

Car park 50 av Maine - 75015 Paris Direct access to Montparnasse station and TGV Tel.: +33 (0)1 43 21 50 30

From Paris airports: The journey from Paris airports to the Institut Pasteur takes from 45 minutes (if you are coming from Orly airport) to 90 minutes (if you are coming from Roissy-Charles de Gaulle airport) Orly airport : Tel.: +33 (0)1 49 75 15 15 Different routes are possible from Orly airport: By Orlyval, you must buy a special ticket to travel between Paris and Orly via Antony which is valid throughout the metro and RER networks. At the Antony RER station, take RER line B to Denfert-Rochereau station. Then take line 6 to Pasteur station.

By Orlybus, you must also buy a special ticket to travel by bus between Paris (Terminus at Denfert-Rochereau) and Orly. From Denfert-Rochereau take the metro (line 6 direction Charles de Gaulle - Etoile) to Pasteur station. You can take the Air France bus to Paris-Montparnasse. You can either walk about 15 minutes or take the metro (line number 6 direction Charles de Gaulle - Etoile) to get from Montparnasse-Bienvenue station to Pasteur station. An Orlyrail shuttle will also take you to the RER C (Aéroport d’Orly station) Finally, the Jetbus will take you to the Villejuif-Aragon metro station, terminus of line 7. Roissy - Charles de Gaulle airport : Tel.: +33 (0)1 48 62 22 80 Different routes are possible from Roissy – Charles de Gaulle airport: Cars Air France (01 41 56 89 00) : CdG - Porte Maillot - Arc de Triomphe CdG - Gare de Lyon - Gare Montparnasse Metro/RER: Go to the RER station at the airport accessible from terminals 1 or 2, or use the free Aéroports de Paris shuttles if your flight lands at the other terminals. Take the RER B to Denfert-Rochereau. Then take line 6 to Pasteur station. By Roissybus This bus goes from the airport to rue Scribe, near place de l’Opéra. Tickets are on sale at line terminus, in the airports and on board buses. By train, from Paris' main SNCF stations: Paris has several SNCF (railway) stations. Below are the routes by metro between the Institut Pasteur and the main stations. “Montparnasse” station This is the closest station to the Institut Pasteur. You can either walk about 15 minutes or take line 6 to Pasteur station. “Saint Lazare” station Take line 12 to Pasteur station (about 20 minutes). “Gare de Lyon” station Take line 14 to Bercy station. Change to line 6 to Pasteur station. Journey time is roughly 35 minutes. “Austerlitz” station Take line 5 to Place d’Italie. Change to line 6 to Pasteur station. Journey time is roughly 30 minutes. “Gare de l’Est” station Take line 4 to Raspail station. Then change to line 6 to Pasteur station. Journey time is roughly 35 minutes. “Gare du Nord” station Take the RER B to Denfert-Rochereau. Change and take line 6 to Pasteur station. Journey time is roughly 35 minutes.

Campus map

Agenda Monday, September 26th (Amphithéâtre Duclaux) Meeting Opens 08:00-08:45 Registration and poster installation 08:45-09:00 Opening remarks by the host and chair of ARBRE MOBIEU,

Patrick England, Institut Pasteur, Paris, France Morning Session 1 Interaction analysis: The value of orthogonal approaches Chair: Patrick England, Institut Pasteur, Paris, France 09:00-09:30 "The Power of Orthogonal Biophysical Methods Exemplified by An Integrative

Structural Biology Study of FGFR1" Geoff Holdgate, AstraZeneca, Cambridge, UK

09:30-09:50 "Combining isothermal titration calorimetry, fluorescence anisotropy and kinetic approaches to decipher the molecular mechanisms of the redox activation of the Yap1 transcription factor in S. cerevisiae" Sophie Rahuel-Clermont, Université de Lorraine, Nancy, France

09:50-10:10 "What have we learned from the enthalpy database of 200 compound binding to 8 carbonic anhydrase isoforms as determined by isothermal titration calorimetry" Daumantas Matulis, Vilnius University, Lithuania

10:10-10:30 Flash presentation of posters (1 slide & 2min/talk) 9 speakers : Regina Adao, University of Campinas, Sao Paulo, Brazil Mathieu Coinçon, Stockholm University, Sweden Andrea Gohlke, Cancer Research Institute UK Beatson Institute, Glasgow, UK Sergei Kuprin, Swedish Orphan Biovitrum AB, Stockholm, Sweden Vaida Linkuviene, Vilnius University, Lithuania Anna Mårtensson, Chalmers University of Technology, Gothenburg, Sweden Ksenia Maximova , Warsaw University, Poland Serena Rinaldo, Istituto Pasteur-Fondazione Cenci Bolognetti, Rome, Italy Katharina Weickhmann, Goethe University, Frankfurt, Germany

10:30-11:00 Coffee break and 1st poster session Morning Session 2 Focus on DSC: Differential Scanning Calorimetry Chair: Michaela Wimmerova, Brno, Czech Republic 11:00-11:30 "Thermal dissociation and unfolding of insulin and effects of ligand interactions

studied by DSC. Predictability of insulin analogue stability using DSC and correlation with other methods" Kasper Huus, Novo Nordisk, Denmark

11:30-11:50 "Study of rabies virus by Differential Scanning Calorimetry: Identification of Proteins Involved in Thermal Transitions" Frederic Greco, Sanofi Pasteur, Marcy l'Etoile, France

11:50-12:10 "Using Differential Scanning Calorimetry to monitor glioblastoma evolution" François Devred, Université Aix-Marseille, France

12:10-12:30 "DSC in application to the studies of blood serum samples" Adrian Velazquez Campoy, Zaragoza University, Spain

12:30-13:30 Lunch 13:30-14:15 Panel discussion 1

"Best practices in DSC experimental setup and data analysis" Chaired by Margarida Bastos, Porto University, Portugal and Natalia Markova, Malvern Instruments, UK

Afternoon session 1 Focus on ITC: Isothermal Titration Calorimetry Chair: Chris Johnson, Cambridge, UK 14:15-14:35 "Study of peptide/lipid interactions by ITC and DSC"

Isabel Alves, CBMN, Bordeaux, France 14:35-14:55 "Enthalpic Forces Correlate with the Selectivity of Transthyretin-Stabilizing

Ligands in Human Plasma" Kristoffer Brännström, Umea University, Sweden

14.55-15:15 "Affinity based definition of RNA motifs and strategic protein-RNA mutational design" Cameron Mackereth, IECB, Bordeaux, France

15:15-15:45 Coffee break and 2nd poster session Afternoon session 2 Novel developments in the field of calorimetry Chair: Margarida Bastos, Porto, Portugal 15:45-16:05 "The underestimated potential of isothermal microcalorimetry for

microbiological studies" Olivier Braissant , Basel University, Switzerland

16:05:16:25 "ITC studies of enzyme activity in solution and suspensions" Peter Westh, Roskilde University, Denmark

16:25-16:45 "Use of isothermal titration calorimetry to investigate and identify novel peptide substrates for prolyl carboxypeptidase. A technology comparison" Anne-Marie Lambeir, Antwerp University, Belgium

16:45-17:05 "KinITC – Kinetic Revolution in Drug Discovery" Pascal Zihlmann, Basel University, Switzerland

17:05-17:50 Panel discussion 2 "Challenges and best practices for the application of ITC to the studies of enzyme kinetics" Chaired by Natalia Markova, Malvern Instruments, UK

17:50-19:30 3rd poster session 19:30-21:15 Gala Dinner

Agenda Tuesday, September 27th (Amphithéâtre Duclaux) Morning Session 1 Developments in ITC applications into challenging areas Chair: by Aymeric Audfray, Malvern Instruments Lyon, France 09:00-09:30 "ITC for Structural Glycosciences tools"

Anne Imberty, CERMAV-CNRS, Grenoble, France 09:30-09:50 "ITC as a tool for the characterization of self-assembled systems in organic

solvents" Laurent Bouteiller, Pierre and Marie Curie University, Paris, France

09:50-10:30 Panel discussion 3 "Challenges and best practices for the use of ITC in non-traditional applications" Chaired by Aymeric Audfray, Malvern Instruments Lyon, France

10:30-11:00 Coffee break and 4th poster session Morning Session 2 Binding complexity and data quality with ITC Chair: by Adrian Velazquez-Campoy, Zaragoza, Spain 11:00-11:30 “Any Kd you like”

Joe Coyle, Astex Pharmaceuticals, Cambridge, UK 11:30-11:50 "Improved characterization of supramolecular assemblies by non conventional

Isothermal Titration Calorimetry experiments" David Landy, Université du Littoral, Dunkerque, France

11:50-12:10 "10 good reasons to perform ITC experiments" Eric Ennifar, Strasbourg University, France

12:10-12:55 Panel discussion 4 "Best practices in ITC data analysis : quality optimization, and appraisal of complex binding and kinetics" Chaired by Eva Munoz, AFFINImeter, Santiago de Compostela, Spain, and Adrian Velazquez-Campoy, Zaragoza University, Spain

12:55-13:15 Concluding remarks Patrick England , Institut Pasteur, Paris, France and Natalia Markova, Malvern Instruments, UK

13:15-14:00 Lunch

Afternoon Workshops Tuesday, September 27th Workshops will be run in parallel, with a maximum of 30 participants each 14:00 ITC Workshop

Led by Aymeric Audfray, Malvern Instruments Lyon, France

DSC Workshop Led by Natalia Markova, Malvern Instruments, UK

14:00-14:45 Part 1. "Principles and applications of ITC. Best practices for experimental design and analysis"

Part 1. How DSC works and why to use DSC when alternatives exist? "Principles and best practices of DSC analysis. Data quality in focus" Natalia Markova, Malvern Instruments (30 min)

"Recent applications of DSC in Oxford" Shared by David Staunton, facility manager of the Biochemistry Department’s Molecular Biophysics Suite, Oxford University (15 min) "

14:45-15:15 Coffee break Coffee break

15:15-16:30 Part 2. "Advanced data analysis (including competition assays, enzyme kinetics and binding kinetics). Best practices to ensure quality of ITC data" With the participation of Angel Pineiro, AFFINImeter, Santiago de Compostela, Spain

Part 2. What can we learn from DSC? "Advanced DSC data analysis" Natalia Markova, Malvern Instruments (25 min) "The use of DSC for characterization of thermotropic phase transitions in lipid bilayer membranes" Margarida Bastos, University of Porto, Portugal (25 min) "Profiling and optimization of protein stability with DSC" Natalia Markova, Malvern Instruments (25 min)

16:30 End End

Speakers – Oral Presentation

Isabel ALVES CBMN, Bordeaux France Sept. 26th - 14:15-14:35

Margarida BASTOS Porto University Portugal Sept. 26th - 13:30-14:15

Laurent BOUTELLIER Pierre and Marie Curie University, Paris

France Sept. 27th - 09:30-09:50

Olivier BRAISSANT Basel University Switzerland Sept. 26th - 15:45-16:05

Kristoffer BRÄNNSTRÖM Umea University Sweden Sept. 26th - 14:35-14:55

Joe COYLE Astex Pharmaceuticals, Cambridge

UK Sept. 27th - 11:00-11:30

François DEVRED Université Aix-Marseille France Sept. 26th - 11:50-12:10

Eric ENNIFAR Strasbourg University France Sept. 27th - 11:50-12:10

Frederic GRECO Sanofi Pasteur France Sept. 26th - 11:30-11 :50

Geoff HOLDGATE AstraZeneca, Cambridge UK Sept. 26th - 09:00-09:30

Kasper HUUS Novo Nordisk Denmark Sept. 26th - 11:00-11:30

Anne IMBERTY CERMAV-CNRS, Grenoble

France Sept. 27th - 09:00-09:30

Anne-Marie LAMBEIR Antwerp University Belgium Sept. 26th - 16:25-16:45

David LANDY Université du Littoral, Dunkerque

France Sept. 27th - 11:30-11:50

Cameron MACKERETH IECB, Bordeaux France Sept. 26th - 14:55-15:15

Daumantas MATULIS Vilnius University Lithuania Sept. 26th - 09:50-10:10

Eva MUNOZ AFFINImeter, Santiago de Compostela

Spain Sept. 27th - 12:10-12:55

Angel PINEIRO AFFINImeter, Santiago de Compostela

Spain Sept. 27th - 15:15-16:30

Sophie RAHUEL-CLERMONT Université de Lorraine, Nancy

France Sept. 26th - 09:30-09:50

David STAUNTON Oxford University UK Sept. 27th - 14:30-14:45

Adrian VELAZQUEZ CAMPOY Zaragoza University Spain Sept. 26th - 12:10-12:30 Sept. 27th - 12:10-12:55

Peter WESTH Roskilde University Denmark Sept. 26th - 16:05-16:25

Pascal ZIHLMANN Basel University Switzerland Sept. 26th - 16:45-17:05

Speakers – Flash Poster Presentation

Regina ADAO University of Campinas, Sao Paulo

Brazil Sept. 26th - 10:10-10:30

Mathieu COINÇON Stockholm University Sweden Sept. 26th - 10:10-10:30

Andrea GOHLKE Cancer Research Institute UK Beatson Institute, Glasgow,

UK Sept. 26th - 10:10-10:30

Sergei KUPRIN Swedish Orphan Biovitrum AB, Stockholm

Sweden Sept. 26th - 10:10-10:30

Vaida LINKUVIENE Vilnius University Lithuania Sept. 26th - 10:10-10:30

Anna MÅRTENSSON Chalmers University of Technology, Gothenburg

Sweden Sept. 26th - 10:10-10:30

Ksenia MAXIMOVA Warsaw University Poland Sept. 26th - 10:10-10:30

Serena RINALDO Istituto Pasteur-Fondazione Cenci Bolognetti, Rome

Italy Sept. 26th - 10:10-10:30

Katharina WEICKHMANN Goethe University, Frankfurt Germany Sept. 26th - 10:10-10:30

Scientific Committee

Scientific Organizing Committee :

Isabel ALVES (Bordeaux, France)

Margarida BASTOS (Porto, Portugal)

Patrick ENGLAND (Paris, France)

Eric ENNIFAR (Strasbourg, France)

Chris JOHNSON (Cambridge, UK)

Daumantas MATULIS (Vilnius, Lithuania)

Eva MUNOZ (Santiago de Compostela, Spain)

Adrian VELAZQUEZ-CAMPOY (Zaragoza, Spain)

Michaela WIMMEROVA (Brno, Czech Republic)

On behalf of Malvern-MicroCal :

Aymeric AUDFRAY (Lyon, France)

Natalia MARKOVA (Malvern, UK)

Interaction analysis: The value of an orthogonal approaches

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

The Power of Orthogonal Biophysical Methods Exemplified by an Integrative Structural Biology Study of FGFR1

Geoff Holdgate1

1Principal Scientist, AstraZeneca, Discovery Sciences, Macclesfield, SK10 4TG, United Kingdom,

The application of orthogonal biophysical methods provide a powerful means to understand biological systems in greater detail, faciliating a deeper understanding for chemical intervention in disease processes. Protein kinases remain important targets for drug discovery and understanding the nature of test compound binding and inhibition is a vital component for effective lead optimisation. This presentation will provide an overview of kinases as drug targets, with specific reference to the FGFR system. It will describe the identification of a candidate molecule as well as highlighting how powerful combinations of biophysical methods, in an integrative structural biology approach, can provide fundamental insights into the nature of protein conformational changes, which may be exploited during drug design.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Combining isothermal titration calorimetry, fluorescence anisotropy and kinetic approaches to decipher the molecular mechanisms of the redox activation of the Yap1 transcription factor in S. cerevisiae

Sophie Rahuel-Clermont1, Antoine Bersweiler1, Benoît d’Autréaux2, Hortense Mazon1, Alexandre Kriznik1, Michel B Toledano2

1IMoPA UMR 7365 CNRS-Université de Lorraine Lorraine – Biopôle, campus Biologie-Santé, 9 Avenue de la Forêt de Haye, CS 50184 54505 -Vandoeuvre-lès-Nancy, France, sophie.rahuel@univ-lorraine.fr, +33 383 68 55 41 2Institute for Integrative Biology of the Cell (I2BC), UMR9198, CNRS, CEA-Saclay, Université Paris-Saclay, iBiTecS/SBIGEM, Laboratoire Stress oxydant et Cancer, Bat 142, F-91198 Gif-sur-Yvette Cedex, France

Thiol-peroxidases are antioxidant enzymes that are also associated with peroxide-dependent cell signaling as sensor and relay of the H2O2-mediated signal (1). One of the best documented examples of such a mechanism is the activation of the transcription factor Yap1, a key regulator of the transcriptional peroxide stress response in Saccharomyces cerevisiae, which depends on the formation of intramolecular disulfide bonds catalyzed by the thiol peroxidase Orp1 (2 ; 3). In this mechanism, the redox relay occurs via the oxidation of Orp1 peroxidatic Cys as a sulfenic acid intermediate which reacts with Yap1 to form a mixed disulfide species (3). In addition, the Ybp1 protein has been identified as an essential partner for the activation of Yap1 by Orp1 (4). The intrinsic reactivity of Orp1 sulfenic acid species raises the question of the specificity of Yap1 activation by H2O2/Orp1. To address this question, and to elucidate the role of Ybp1 in this mechanism, we have used an approach combining i) the characterization of the interactions between the protein partners Orp1, Yap1 and Ybp1 by isothermal titration microcalorimetry and fluorescence anisotropy, and ii) the kinetic characterization of the peroxidatic cycle of Orp1 and of its reaction with Yap1, using rapid kinetic techniques. We propose that Ybp1 and Yap1 recruit Orp1 within a ternary complex, which restrains intramolecular disulfide formation within Orp1 and allows the reaction between Orp1 sulfenic intermediate and Yap1. Our study thus highlights the pertinence to combine protein interaction and kinetic approaches to characterize the processes involved in redox signaling pathways.

1. Fourquet S., et al. (2008), Antiox. And redox signaling 10, 15565-76.2. Delaunay, A., et al. (2000) EMBO J. 19, 5157-66.3. Delaunay, A., et al. (2002) Cell 111, 471-81.4. Veal, E.A., et al. (2003) J. Biol. Chem. 278, 30896-904.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

What have we learned from the enthalpy database of 200 compound binding to 8 carbonic anhydrase isoforms as determined by isothermal titration calorimetry Asta Zubrienė, Vaida Linkuvienė, Lina Baranauskienė, Alexey Smirnov, Daumantas Matulis Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University LT-10257, Vilnius, Lithuania. Target-based drug design is based on discovery and selection of a most-strongly binding compound to target protein. However, the binding affinity is the sum of enthalpic and entropic forces determining the binding mechanism that is usually an interplay of highly compensating enthalpic and entropic contributions. Even homologous compounds that exhibit similar affinities may have significantly different enthalpies and entropies of binding. When high-resolution crystallographic structures are available for all compound complexes with the target protein, sometimes it is possible to assign these significant enthalpy and entropy differences to the behavior of the water molecules located at the compound-protein binding interface. We have designed, synthesized and determined the binding thermodynamics of over 700 aromatic sulfonamides to the family of 12 human carbonic anhydrase (CA) isoforms. The proteins were cloned and expressed in bacterial and human cell cultures and affinity-purified in large quantity sufficient for ITC. The binding affinities were determined by the thermal shift assay (also termed ThermoFluor or differential scanning fluorimetry), a high-throughput method. The enthalpies and entropies of binding were determined by ITC, a medium throughput method, for a part of compounds and CA isoforms. A correlation map between the compound chemical structure and the binding ∆G and ∆H was drawn. The map showed which structural features of the compounds generated highest increments in exergonicity and exothermicity of compound binding. Furthermore, only some structural features were most useful in generating compounds that would selectively bind to cancer-expressing CA isoforms, but would not bind to essential for life human CA isoforms. ITC was essential method that enabled us to dissect unknown contributions from linked reactions such as buffer protonation to the binding reaction. Only after the subtraction of pH-dependent buffer contribution to the enthalpy of binding, we obtained the intrinsic Gibbs energies and enthalpies of binding. All methods that determine the binding reaction, such as the thermal shift assay, ITC, SPR, thermophoresis and enzymatic inhibition methods would provide only the observed thermodynamics of binding that is pH and buffer-dependent. It was important to calculate the true (intrinsic) parameters and use them in the structure-thermodynamics correlation maps that will be discussed at the conference

Flash presentation of poster

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Thermal stability and interaction of heat shock protein Hsp90 with its TPR co-chaperones

Regina Adão1, Conrado C. Gonçalves2, Letícia M. Zanphorlin3, Carlos H.I.Ramos4

1-4Institute of Chemistry, University of Campinas - UNICAMP, São Paulo, Brazil, 1reginaca@iqm.unicamp.br;2conradodecampos@gmail.com; 3lmzanfa@yahoo.com.br; 4cramos@iqm.unicamp.br

Incorrect protein folding into cells can cause several conformational diseases and in order to prevent such structural incorrectness, cells have evolved an efficient protein quality control system (PQC) as a housekeeping process to maintain homeostasis. Molecular chaperones (including all HSP families) are essential to the PQC system and their main function is to prevent inappropriate interactions, avoiding protein aggregation by assisting their correct folding or guiding them to cell degradation system1-3. One of the most important chaperones is the Heat-shock protein 90 kDa (HSP90), which is responsible for the correct folding of a wide range of client proteins4,5. In the folding process, it is essential that HSP90 form complexes with co-chaperones containing TPR domains, providing a cooperative action during the maturation cycle of client proteins. Co-chaperones CHIP, HOP and TOM are examples of TPR proteins that interact with HSP90 in order to assist it in many functions. CHIP, an E3 ubiquitin-ligase, is responsible to maintaining the balance between correct folding and targeting proteins to the proteasome. HOP is an adapter modulating substrate delivery to Hsp90, by binding to the MEEVD motif of HSP90 C-terminal domain. TOM is a mitochondrial intermembrane protein that receives the newly synthesized protein from the ribosome (pre-protein), forming a complex-TOM-HSP90-pre-protein able to move through mitochondrial channel6-10. In this work, we study the thermal stability of the chaperone (Hsp90) and co-chaperones CHIP, HOP and TOM by DSC. Hsp90-CHIP, Hsp90-HOP and Hsp90-TOM interactions were performed by ITC in order to unravel the structural and conformational basis of the processes in which chaperones interact with their substrates.

HSP: heat shock protein; TPR: tetratricopeptide repeat; CHIP: carboxyl-terminus of Hsc70-interacting protein; HOP: heat shock organizing protein; TOM: translocase of the mitochondria outer membrane.

1. Tiroli-Cepeda, A.O., Ramos, C.H. Protein & Peptide Letters (2011), 18(2): p. 101-109.2. Walter, S. and J. Buchner. Angewandte Chemie International Edition (2002), 41(7): p. 1098-1113.3. Hartl, F.U., A. Bracher, and M. Hayer-Hartl., Nature (2011), 475(7356): p. 324-332.4. Buchner, J. Trends in Biochemical Sciences (1999), 24(4): p. 136-141.5. Da Silva, V.C.H. and C.H.I. Ramos. Journal of Proteomics (2012), 75(10): p. 2790-2802.6. Allan, R.K. and T. Ratajczak. Cell Stress & Chaperones (2011), 16(4): p. 353-367.7. Wandinger, S.K., K. Richter, and J. Buchner. Journal of Biological Chemistry (2008), 283(27): p. 18473-18477.8. McDonough, H. and C. Patterson. Cell Stress & Chaperones (2003). 8(4): p. 303-308.9. Goncalves, Danieli C., Gava, Lisandra M., Ramos, Carlos H.I. Protein and Peptide Letters (2010), 17(4): p. 492-

498.10. Gava, L.M., Gonçalves, D.C., Borges, J.C., Ramos, C,H. Archives of Biochemistry and Biophysics (2011), 513: p.

119–125.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

ITC measurement of Na+ and Li+ binding in the Sodium/proton exchangers NapA

Mathieu Coinçon1, David Drew2

1Stockholm University, DBB Svante Arrhenius väg 16C 10691 Stockholm Sweden, mathieu.coincon@dbb.su.se, +46 722652977 2david.drew@dbb.su.se

Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels and cell volume. In humans, their dysfunction has been linked to a number of diseases such as hypertension and cardiac pathologies. For elucidating the transport mechanism of NapA from Thermus thermophilus we had to engineer a dispulphide bond in our protein in order to trap the missing conformation of this transporter. We verified that these mutations did not impact the transport of the substrate across proteolyposomes and we solve its structur. But we also needed to check that the disulphide-trapped structure was physiologically relevant so was still able to bind Na+. While ITC measurement with the wild-type protein failed (our protein express poorly, the substrate binding is weak and detergent requirement added another degree of complexity to the measurement), use of the trapped protein was successfull. Indeed, after a careful sample preparation and choice of a suitable buffer we were able to titrate Na+ binding to a Sodium/proton exchangers for the first time and to check its pH dependency.

Coincon, M. et al. Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters. (2016) Nat. Struct. Mol. Biol. 23, 248–255 (2016)

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Inhibition of the fascin-actin interaction using a multidisciplinary fragment-based screening approach

Andrea Gohlke1, Daniel Croft1, Stuart Francis1, Alexander Schuettelkopf1, Charles Parry1, Gillian Goodwin1, Angelo Pugliese1, Laura McDonald1, Peter Nicholas Brown1, Nikki Paul2, Sophie Macconnachie1, Andrew Pannifer1, Jen Konczal1, Christopher Gray1, Justin Bower1, Heather McKinnon1, Laura Machesky2 and Martin Drysdale1

1Drug Discovery, Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; 2Migration, Invasion & Metastasis Laboratory, Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; A.Gohlke@beatson.gla.ac.uk

Fascin 1 is an actin bundling protein contributing to the evolvement of dynamic cellular protrusions used during cell migration. Its expression is low in normal epithelia but is increased in a variety of metastatic tumors. Here, it has been reported to contribute to the formation of actin-rich protrusions which are involved in the degradation of the extracellular matrix. Fascin 1 is currently used as a prognostic indicator of poor clinical outcome. Its knockdown reduces tumor invasion, proposing it as a valid drug target. However, as the detailed interaction of fascin 1 with actin is still unclear it remains a highly challenging target. Using a multidisciplinary approach of fragment-based screening we were successful in identifying novel sub-micromolar fascin 1 inhibitors. Initially, from a surface plasmon resonance screen of 1000 fragments 53 fascin1 binding hits were identified. Using co-crystallography we distinguished four ligand binding sites. Focussing on a deeply enclosed pocket between fascin domains 1/ 2, fragment hits and analogues were found to open a channel towards the protein surface. Virtual screening finally identified a compound which binds with an SPR Kd of 30 µM and an IC50 of 50 µM as revealed in a functional biochemical screening assay that measures fascin-mediated actin bundling. After structure-based optimization and co-crystallography compounds with > 100 fold increase in both binding affinity and functional activity were identified. Following this, we are currently working on the optimisation of these compounds exhibiting sub-micromolar affinity using isothermal calorimetry and fluorescence polarisation as additional biophysical methods.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Coupling of Stability and Self-Association of a Therapeutic Protein

Sergei Kuprin1, Erik Nordling1, Vilhelm Ek11, Natalia Markova2 1Swedish Orphan Biovitrum AB, 11276, Stockholm, Sweden, sergei.kuprin@sobi.com, +46767222065, 2Malvern Instruments Ltd, natalia.markova@malvern.com

Construct 50 is a small, 2-domain recombinant protein candidate drug. Only limited conclusions on protein stability have been obtained with optical methods. In SEC elution was dependent on the column load thus indicating self-association. An ITC and DSC study was initiated in attempt to understand protein behavior and stability. Measurements were made on MicroCal TMAuto VP Capillary DSC system and on MicroCalTM iTC200 at GE Healthcare DemoLab (at present: Malvern Instruments Ltd Demo Lab in Stockholm). DSC revealed two distinct thermal transitions: Tm1 ~27-37 °C and Tm2 ~55-70 °C. Parameters varied with ionic strength of the buffer. The first transition was strongly dependent on protein concentration, while the second one was much less affected. The first transition can be coupled to self-association of the protein, that was favored by low temperatures and low ionic strength. Were also studied twelve other genetic variants. The constructs varying in one or a few amino acid positions yielded significantly different DSC thermograms. Again two transitions were identified and most variability was observed for the first transition. In ITC experiments concentrated protein was diluted in a stepwise manner with a respective buffer. The data confirmed temperature and buffer dependent oligomerization of the protein and correlate with SEC. Conclusions: DSC revealed conditions of maximal structuring coupled to oligomerization. The knowledge is important for development of therapeutic formulations. ITC allowed measurements of equilibrium constants of oligomerization, which is important for assessment of drug behavior in solutions and in human body)

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Comparison of titration calorimeters : High affinity and enthalpy precision

Vaida Linkuvienė, Daumantas Matulis

Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, 10257, Vilnius, Lithuania, morkunaite@ibt.lt, matulis@ibt.lt.

Isothermal titration calorimetry (ITC) is one of the most robust label- and immobilization-free techniques used to measure the affinity of interaction between two molecules that are both sufficiently soluble in aqueous solution. ITC is most often used to measure protein – small molecule interaction in drug design. Furthermore, ITC is an indispensable technique that directly determines the enthalpy of interaction – an important thermodynamic parameter, largely underused in research. ITC data, both the affinity (Gibbs energy) and the heat of reaction (enthalpy, when pressure = const), are highly useful if the experiments are performed properly. Here we compare data obtained by five ITC calorimeters, namely, PEAQ-ITC, ITC200, VP-ITC, Nano-ITC and MCS-ITC, using the same reaction, a convenient interaction between carbonic anhydrase II (CA II) and acetazolamide (AZM). Furthermore, limitations and the precision of the determination of affinity and enthalpy were shown using other high-affinity reactions. The new PEAQ-ITC calorimeter was found to be able to achieve the most precise determination of the tight affinity that is often beyond ITC reach by performing direct (non-displacement) ITC experiment.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Heterochiral cooperativity or not?

Anna K. F. Mårtensson1 and Per Lincoln1

1 Physical Chemistry, Dept of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden, marann@chalmers.se, lincoln@chalmers.se

DNA intercalation is a non-covalent binding mode where the DNA helix is unwound in order for the intercalating ligand to π-stack between two base pairs. This type of binding has played an essential part in the development of new pharmacological therapeutics and biotechnological applications. Intercalating ruthenium complexes can either be chiral, with the enantiomers named delta (Δ) for a right-handed, and lambda (Λ) for a left-handed propeller sense, or achiral, where the metal is lacking a stereocenter. As the DNA-strand most commonly is a right-handed helix, its interactions with chiral ruthenium complexes will be diastereomeric.1 In this study, we have explored the nearest-neighbour interactions between consecutive complexes when intercalated to a DNA polymer using calorimetric measurements. We created a competitive setting where the second complex had an opportunity to replace another complex already intercalated to DNA. Both Ru(bpy)2dppz2+and Ru(tpy)(py)dppz2+ were included in this study as they represent the basic structures of chiral and achiral intercalating ruthenium complexes.1,2 Using a McGhee and von Hippel type of method to calculate the binding isotherms,3 we were able to find a binding model with an excellent global fit for the intercalated complexes. By developing simulated binding models it will be possible to predict DNA-complex interactions. This will be an important aid in the development of new, more efficient metallo-pharmaceuticals.

1. J. Andersson, et al., Inorg. Chem., 2013, 52(2): p. 1151-11592. A. K. F. Mårtensson, et al., Dal. Trans., 2015, 44(8): p. 3604-36133. P. Lincoln, Chem. Phys. Let., 1998, 288(5-6): p. 647-656

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Isothermal titration calorimetry for measuring enzyme kinetics and inhibition.

Ksenia Maximova1, Joanna Trylska1 1Centre of New Technologies University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland, k.maximova@cent.uw.edu.pl

Up to now, ITC has been commonly applied to determine the thermodynamic parameters of association of molecules and has not been appreciated to investigate chemical reactions catalyzed by enzymes. Our aim was to apply ITC for various types of trypsin catalyzed reactions to figure out the benefits and limitations of this technique for enzyme kinetic analysis [1]. First, the amide hydrolysis was assayed with casein as a substrate. Since casein is an insoluble macromolecule substrate, it allowed us to examine the ITC technique for turbid solutions. Second, the ester hydrolysis was monitored with Nα-benzoyl-DL-arginine β-naphthylamide (BANA) as a substrate. This small BANA substrate contains a fluorescent naphthylamine group, which, in addition, allowed us to compare the efficiency of ITC with a classical fluorometric enzymatic assay. Furthermore, we explored the potential of ITC in estimating the inhibition of the reaction by its products and different types of inhibitors. We used both reversible (benzamidine) and irreversible (phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin to estimate the inhibition constants. Our experiments showed that ITC is an accurate, fast and straightforward method for the determination of kinetic constants, as well as enzyme inhibition. Overall, ITC is a powerful method to study enzyme kinetics and inhibition for complex solutions and natural substrates.

1. Maximova, K., and Trylska, J. (2015) Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titrationcalorimetry. Anal. Biochem. 486, 24–34.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Unselective Bimetallic Binding Site of the Pseudomonas aeruginosa PA4781 Protein HD-GYP Domain Revealed by ITC: a novel class of sensors in controlling biofilm formation?

Serena Rinaldo1, Alessandro Paiardini2, Valentina Stelitano1, Paolo Brunotti1, Laura Cervoni1, Silvia Fernicola1, Matteo Vitali3, Francesca Cutruzzolà1, Giorgio Giardina1

1Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences; 2Department of Biology and Biotechnology “Charles Darwin”; 3Department of Public Health and Infectiuos Diseases, Sapienza University of Rome (Italy), serena.rinaldo@uniroma1.it

The intracellular level of the bacterial secondary messenger cyclic di-(3',5')-guanosine monophosphate (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs), bearing characteristic EAL or HD-GYP domains (1). We have obtained the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously functionally characterized in vitro (1,2). The structure shows a bi-metallic active site whose metal binding mode is different from both HD-GYP PDEs structurally characterized so far (2-4). Purified PA4781 does not contain iron in the active site as for other HD-GYPs (3-5), and we show by ITC that it binds to a wide range of transition metals with similar affinity. ITC data underlined that the metal binding event is allosterically controlled and gated by the protein moiety (2). Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested (1). Our results point out that the structural features of HD-GYPs are more complex than predicted so far, and identify the HD-GYP domain as a conserved scaffold, evolved to preferentially interact with a partner GGDEF, but harbouring different functions obtained through diversification of the active site (2).

1. Stelitano, V., et al. (2013) PLoS One 8, e74920.2. Rinaldo, S., et al. (2015) J Bacteriol 197, 1525-35.3. Lovering, L.A., et al. (2011) MBio 2, 1–8.4. Bellini, D., et al. (2014) Mol. Microbiol. 91, 26–38.5. Miner, K.D., et al. (2013) Biochemistry 52, 5329–31.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

The ligand binding mode and a protonated adenine of a GTP-aptamer A. Katharina Weickhmann1, Antje C. Wolter1, Elke Duchardt-Ferner1, Katharina M. Hantke1, Amir H. Nasiri1 and Jens Wöhnert1 1Institute for Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt, Germany Aptamers are single-stranded nucleic acids that are selected in vitro for their high affinity binding to a specific target molecule. The GTP class II aptamer binds GTP with an affinity in the nanomolar range and it recognizes the ligand mainly via its Watson-Crick (WC) face [1]. Using ITC and NMR spectroscopy, we were able to characterize the ligand binding mode and showed that the sugar pucker and the phosphate groups are not recognized by the aptamer. Interestingly, when we compared ITC measurements at high and low magnesium concentrations, we found that the strong negative charge of the ligand bound to an also negatively charged RNA is compensated by magnesium ions. We solved the structure of the RNA-ligand bound complex by solution NMR. The structure revealed that GTP is bound by a WC base pair to a conserved cytidine residue and a sugar edge interaction. At the base of the ligand binding site we found a base quadruplet with a stably N1 protonated adenine. Using pH dependent ITC measurements, we were able to determine the pKa of the free and the ligand-bound aptamer which is highly shifted compared to free adenine. Interestingly, the stable protonation was destabilized by inserting an additional adenine at a flexible linker of the aptamer which we analyzed by ITC. Overall, the GTP class II binding aptamer shows many interesting structural features which we successfully characterized by a combination of NMR spectroscopy and ITC measurements. 1. Carothers, J. M.; Oestreich, S. C.; Davis, J. H.; Szostak, J. W. J. Am. Chem. Soc., 2004, 126, 5130-5137

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Thermodynamics of Oppositely Charged Biopolymers

Aysun Bulut1, Fatma Akcay Ogur1, A.Basak Kayitmazer1,

1Department of Chemistry, Bogazici University aysunbulutbulut@gmail.com, 2Department of Chemsitry, Bogazici University, fatma.akcay@boun.edu.tr.

Coacervation is a phenomenon in which a macromolecular aqueous solution separates into two immiscible liquid phases.This liquid-liquid phase separation technique has been extensively used in cosmetic formulations and pharmaceutical microencapsulations. Isothermal titration calorimetry (ITC) has been widely used for the characterization of polymer/ligand and complexes between oppositely charged species. ITC is now routinely used to directly characterize the thermodynamics of biopolymer binding interactions. [3-4]. Our purpose here is to use ITC to increase our understanding of the complex formation between oppositely charged hyaluronic acid (HA) and chitosan (CH). The interaction between CH and HA was studied by ITC to obtain information about binding energetics, thermodynamics and stoichiometry of the interactions. All experiments were performed with a GE Healthcare (Microcal) VP-ITC. The experiments were carried out at different ionic strengths to determine the effect of salt concentration on the binding process. At constant salt concentration, change in the ratio of HA to CH followed a non-monotonic behaviour. At constant HA to CH ratio (by mole), increase in salt concentration resulted as lower binding constants indicating the effect of charge screening. Positive entropy values indicate that the interaction is entropically driven due to counterion release. Positive enthalpy values indicate that the interaction is endothermic. In summary, HA-CH interaction is enthalpically and entropically driven.

1. A. Veis A review of the early development of the thermodynamics of the complex coacervation phase separationAdvances in Colloid and Interface Science 167 (2011) 2–11

2. Wang et al Effects of Salt on Polyelectrolyte-Micelle Coacervation Macromolecules, Vol. 32, No. 21, 19993. V.H. Le et al. Modeling complex equilibria in isothermal titration calorimetry experiments: Thermodynamic

parameters estimation for a three-binding-site model Anal. Biochem. 434 (2013) 233–2414. Dimitrios Priftis, Nicolas Laugel and Matthew Tirrell Thermodynamic Characterization of Polypeptide Complex

Coacervation, American Chemical Society, Langmuir 2012, 28, 15947−15957

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand Murielle JAM1, Elizabeth Ficko-Blean, Aurore Labourel, Robert Larocque, Mirjam Czjzek and Gurvan Michel

1Sorbonne Université, UPMC Univ Paris 06, CNRS, Integrative Biology of Marine Models, Station Biologique, jam@sb-roscoff.fr

Laminarin is an abundant brown algal storage polysaccharide. Marine microorganisms, such as Zobellia galactanivorans, produce laminarinases for its degradation, which are important for the processing of this organic matter in the ocean carbon cycle. These laminarinases are often modular, as is the case with ZgLamC which has an N-terminal GH16 module, a central family 6 carbohydrate-binding module (CBM) and a C-terminal PorSS module. To date, no studies have characterized a true marine laminarin-binding CBM6 with its natural carbohydrate ligand. The crystal structure of ZgLamCCBM6 indicates that this CBM has two clefts for binding sugar (variable loop site, VLS; and concave face site, CFS). The ZgLamCCBM6 VLS binds in an exo-manner and the CFS interacts in an endo-manner with laminarin. Isothermal titration calorimetry (ITC) experiments on native and mutant ZgLamCCBM6 confirm that these binding sites have different modes of recognition for laminarin, in agreement with the ‘regional model’ postulated for CBM6-binding modules. Based on ITC data and structural data, we propose a model of ZgLamCCBM6 interacting with different chains of laminarin in a multivalent manner, forming a complex cross-linked protein–polysaccharide network. 1. JAM et al. (2016) The FEBS Journal

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Molecular approach of the synergistic effect on astringency elicited by mixtures of flavanols

Alba María Ramos-Pineda1, Montserrat Dueñas1, Julián Rivas-Gonzalo1, Ignacio García-Estévez1,2, María Teresa Escribano Bailón1

1Grupo de Investigación en Polifenoles (GIP), University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca (Spain), 2LAQV, REQUIMTE, Universidade do Porto, Porto, Portugal, amramos@usal.es, mduenas@usal.es, jcrivas@usal.es, igarest@usal.es, escriban@usal.es

Flavan-3-ols are a large and complex group of compounds commonly found in plants, food and beverages. These compounds can interact with salivary proteins, which in turn is the main mechanism for development of astringency sensation. Most of the studies devoted to the interaction protein-polyphenol have been performed using commercial proteins like bovine serum albumin (BSA) or alpha-amylase; but studies with human salivary proteins are scarce. Recently we have evidenced, by sensory evaluation, the existence of synergisms between phenolic compounds on the astringency they elicit [1]. With the aim to study the synergistic effect between flavan-3-ols and human salivary proteins, we have evaluated and compared the interaction between flavan-3-ols, (+)-catechin (CAT) and (-)-epicatechin (EC), and human salivary proteins, individually and as a mixture of both (1:1), maintaining constant the final concentration, using HPLC-DAD and isothermal titration calorimetry (ITC). Thermodynamic parameters obtained from ITC data provided us information about the molecular mechanism of this interaction.

1. Ferrer-Gallego, R. et al. (2014) Food Research International 62, 1100-1107.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Conformational and solvation entropy differences in Galectin-3 upon binding diastereomeric pair of designed ligands

Olof Stenström,1 Francesco Manzoni,2 Maria Luisa Verteramo,3 Majda Misini Ignjatovic,4 Martin Olsson,4 Hakon Leffler,5 Ulf Ryde,4 Derek Logan,2 Ulf J. Nilsson3 and Mikael Akke2

1Center for Molecular Protein Science, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Sweden, olof.stenstrom@bpc.lu.se, 2Center for Molecular Protein Science, Department of Chemistry, Lund University, P.O. Box 124, SE-22100, Sweden, 3Center for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden, 4Theoretical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden, 5Department of Laboratory Medicine, Lund University, P.O. Box 117, SE-22100, Sweden

Conformational fluctuations of proteins are potentially important in ligand binding1,2, but often disregarded in drug design. Here, we have probed the conformational entropy of Galectin-3 upon binding two designed ligands, and also estimated the solvation enthalpy and entropy of the binding process, using a combination of ITC, X-ray crystallography, NMR relaxation, and computational methods. Galectin-3 is a β-galactoside binding lectin3, with important roles as a mediator of physiological responses, such as inflammation4,5 and immunity6. We used a pair of ligands with minimal structural differences, limited to their stereochemistry. The results reveal the importance of conformational entropy in ligand binding and demonstrate entropy-enthalpy compensation between the complexes.

1. Diehl, C.; Genheden, S.; Modig, K.; Ryde, U.; Akke, M. Conformational Entropy Changes upon Lactose Binding tothe Carbohydrate Recognition Domain of Galectin-3. J Biomol NMR 2009, 45 (1-2), 157–169.

2. Akke, M. Conformational Dynamics and Thermodynamics of Protein-Ligand Binding Studied by NMR Relaxation.Biochem Soc Trans 2012, 40 (2), 419–423.

3. Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y.; Poirier, F. Introduction to Galectins. Glycoconjugate J. 2004, 19,433–440.

4. Henderson, N. C.; Sethi, T. The Regulation of Inflammation by Galectin-3. Immunol. Rev. 2009, 230, 160–171.5. Rabinovich, G. A.; Baum, L. A.; Tinari, N.; Paganelli, R.; Natoli, C.; Liu, F.-T.; Lacibelli, S. Galectins and Their

Ligands: Amplifiers, Silencers or Tuners of the Inflammatory Resonse? TRENDS Immunol. 2002, 23 (6), 313–320.6. Yang, R.-Y.; Hsu, D. K.; Liu, F.-T. Expression of Galectin-3 Modulates T-Cell Growth and Apoptosis. Proc. Natl.

Acad. Sci. 1996, 93, 6737–6742.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

An Evaluation of Drug Binding Characteristics of Drug-Eluting Embolisation Beads using Isothermal Titration Calorimetry

Tanya S. Swaine1,2, Gareth Parkes1, Yiqing Tang2, Pedro Garcia2, Laura Waters1, Andrew L. Lewis2

1Tanya Swaine, Tanya.Swaine@hud.ac.uk

Isothermal titration calorimetry (ITC) was able to identify the binding ratio of a drug eluting bead (DEB). Precise amounts of doxorubicin (dox) were titrated into polymer beads (DC Bead™, a device used in performing transarterial chemoembolisation (TACE)). Similar results were found to that of reported UV analysis with additional information collected [1]. In a previous study no difference was seen between the binding ratios at temperatures of 20-40 °C [2], however with further investigation temperature was found to increase the rate of binding. Concentration had an effect on how the ligand bound to the excipient, yet there was no significant difference between the ratios of the reactant bound to the macromolecule. The volume and number of injections did alter the binding ratio, however this was due to the error associated with high and low volumes of injectant. The binding site of the bead is 2-acrylamido-2-methylpropane sulfonate sodium salts (AMPS); the same amount of AMPS from a 50 % v/v solution of AMPS in water was required to achieve a profile similar to that of dox into beads. UV analysis of the residual solution from the ITC sample and forced extraction of the drug from the beads following the experiments displayed comparable results to the ITC, whereby related amounts of drug were identified in the saturated beads and remained in the surplus solution.

1. Gonzalez, M.V., Delivery of Drugs from Embolisation Microspheres. 2006, University of Brighton.2. Waters, L.J., T.S. Swaine, and A.L. Lewis, A calorimetric investigation of doxorubicin-polymer bead interactions.

Int J Pharm, 2015. 493(1-2): p. 129-33.

Focus on DSC: Differential Scanning Calorimetry

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Insulin thermostability, ligand interactions, and correlation of DSC data with other stability indicating methods Kasper Huus1

1 Peptide and Protein Formulation, Injectable Drug Product, Global Research, Novo Nordisk A/S.

In order to fully benefit from using DSC for studying variations in protein mutations and formulation compositions, the thermal denaturation mechanism should be understood. The thermal stability of human insulin has been studied by DSC and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at ∼70 °C. The two monomeric insulin mutants AspB28 and AspB9,GluB27 unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with Tm’s of ∼70 and ∼87 °C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (≥5 Zn2+/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. With the understanding of the thermal denaturation mechanism, we have studied the correlation between thermal denaturation and other stability indicating methods such as chemical and physical stability. It was found that DSC can be used to predict B3 deamidation in human insulin. In a series of novel insulin analogues we found a good correlation between DSC data and covalent dimerization. In general, we have found that DSC is an excellent tool for prediction of stability insulin analogues, especially when insulin is formulated to be in the stable hexamer configuration.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Study of rabies virus by Differential Scanning Calorimetry: Identification of Proteins Involved in Thermal Transitions1 A.Toinon, F. Greco, N. Moreno, M.C. Nicolaï, C. Manin, F.Guinet-Morlot &F. Ronzon

Sanofi Pasteur, Campus Mérieux, 1541 Avenue Marcel Mérieux, Bâtiment X3, 69280 Marcy l'Etoile, France, corresponding author : catherine.manin@sanofipasteur.com

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.

1. Toinon et al. (2015) Biochemistry and Biophysics Reports 4 (2015) 329-336

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

DSC in application to the studies of blood serum samples Adrian Velazquez-Campoy1, Sonia Vega2, Olga Abian3.

1Institute BIFI, University of Zaragoza, Zaragoza, Spain; Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain; Fundacion ARAID, Zaragoza, Spain. adrianvc@unizar.es, 2 Institute BIFI, University of Zaragoza, Zaragoza, Spain. svega@bifi.es, 3 Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain; Institute BIFI, University of Zaragoza, Zaragoza, Spain. oabifra@unizar.es

Traditionally, Differential Scanning Calorimetry (DSC) has been employed for characterizing thermally-induced conformational transitions of biomolecules; in particular, the thermodynamic parameters of protein thermal denaturation and the stabilizing effect of ligand binding. Chaires et al. proposed this technique as a potential tool for disease diagnosis and monitoring through the analysis of blood serum from patients1-4. Since then, many works have contributed to the validation of DSC as a potential quick, non-invasive tool for diagnosing and discriminating several malignancies. The underlying hypotheses in applying DSC in clinical diagnosis are: 1) the thermogram acquired from the thermal denaturation reflects the complex protein and metabolite composition/interaction of the plasma sample (metabolites may not undergo conformational transitions, but they can interact with proteins, modulating their thermal stability), or “interactome hypothesis”; and 2) pathologies and disorders trigger alterations in protein and metabolite composition in plasma (up- or down-regulation of specific proteins and the presence/absence of metabolites specifically related to the disease), which will be mirrored in distorted thermally-induced conformational transitions and, therefore, distorted thermograms when compared to those from healthy subjects. One of the main advantages of using DSC with plasma samples is that a minimally invasive assay such as a routine blood test could help to: 1) diagnose the disease at an early stage; 2) monitor the remission of the disease or relapse in treated patients; and 3) anticipate the decision-making process during medical treatment by predicting theevolution of the disease.Results obtained from different patients groups, in particular gastric adenocarcinomapatients5, will be presented.

1. Garbett N.C., et al. (2007) Clin Chem 53, 2012-20142. Garbett N.C. et al. (2007) Semin Nephrol 27, 621-6263. Garbett N.C. et al. (2008) Biophys J 94, 1377-13834. Garbett N.C. et al. (2009) Exp Mol Pathol 86, 186-1915. Vega S. et al. (2013) Sci Rep 5, 7988

Focus on ITC: Isothermal Titration Calorimetry

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Study of peptide/lipid interactions by ITC and DSC

Astrid Walrant1, Cherine Bechara1, Sandrine Sagan1 and Isabel Alves2

1 Laboratoire des Biomolecules, Universite Pierre et Marie Curie, UMR 7203 CNRS-ENS, cc182, 4 place Jussieu, 75252 Paris cedex 05, France; 2Chimie et Biologie des Membranes et Nano-objets, UMR 5248 CNRS, Allée Geoffroy St. Hilaire, 33600 Pessac, France

The characterization of peptide/lipid (P/L) interactions is detrimental to elucidate the mechanisms of action of membrane active peptides such as cell penetrating, antimicrobial, viral and amyloid peptides. Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) provide a complete thermodynamic characterization of the interaction of such peptides with lipid model systems of varied lipid composition and thus greatly contribute to the understanding of their action mode. The present work will focus in the characterization of the interaction of cell penetrating peptides (CPPs) with lipid model systems (unilamellar vesicles and multilamelar vesicles) and with whole cells. CPPs are very promising therapeutic molecules as they are able to cross cell membranes and transport cargos of varied size and nature inside cells. Despite their potential, the mechanism of action of such peptides remains obscure. The cell membrane constitutes the first barrier the peptides encounter, thus understanding P/L interactions is essential. The present studies have demonstrated the importance of electrostatic interactions between the peptide and the lipids and the capacity of peptides to recruit anionic lipids from binary lipid mixtures.1,2 The importance of carbohydrates for P/L interactions is also elucidated by measuring the affinity of the peptides for cell lines that express different types of carbohydrates.3 Finally, the interaction of CPPs possessing varied number of Trp residues with different glycosaminoglycans (GAGs) is monitored by ITC in a way to investigate the role of Trp residues and that of GAGs in P/L interaction.4

1. I. D. Alves et al (2008). Biochim. Biophys. Acta 1780, 948-592. P. Joanne et al (2009) Biochim. Biophys. Acta 1788, 1772-17813. I. D. Alves et al (2011) PLoS One 6(9):e24096.4. C. Bechara et al (2012) FASEB J. 27, 738-49.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Enthalpic Forces Correlate with the Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma

Brännström K1, Iakovleva I1, Nilsson L2, Gharibyan AL3, Begum A2, Sauer-Eriksson AE2, Olofsson A1.

1Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden, Kristoffer.brannstrom@umu.se, Irina.iakovleva@umu.se, anders.olofsson@umu.se, 2Department of Chemistry, Umeå University, 901 87 Umeå, Sweden, lina.nilsson@umu.se, afshan.begum@chem.umu.se, elisabeth.sauer-eriksson@chem.umu.se, 3Department of Pharmacology and Clinical Neurosciences, Umeå University, 901 87 Umeå, Sweden, anna.gharibyan@umu.se, 4Department of Public Health and Clinical Medicine, Umeå University , 901 87 Umeå, Sweden

The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Affinity based definition of RNA motifs and strategic protein-RNA mutational design Cameron Mackereth,1 Samir Amrane,1 Heddy Soufari,1 Denis Dupuy1

1 Inserm U1212, Univ. Bordeaux, Institut Europeen de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac, France c.mackereth@iecb.u-bordeaux.fr, +33 (0) 5 40 00 34 32

The interaction between RNA-binding proteins and their RNA target sequences is a key component of the production of the final mRNA. In particular, protein-RNA interactions are critical to define the intron sequences that must be removed in normal splicing, although additional RNA-binding proteins can also bind in a regulatory manner to change the splicing patterns in specific tissues or in response to external stimuli. We have used isothermal titration calorimetry (ITC) coupled with structural investigation to understand the RNA motifs bound by several splicing factors, by using extensive series of ligands with sequence variation in each position to determine the optimal high affinity RNA sequences.1,2 Following the generation of structural models, we again rely on ITC to probe all key elements in the recognition of the RNA by specific residues by using a new technique of pairwise mutational study. This validation combines both RNA and protein mutation with ITC measurements to precisely identify critical protein-RNA contacts at the level of specific atoms. Finally, we also look at cooperative effects between different splicing factors that bind to adjacent positions on the mRNA, in order to understand at the molecular level the interplay of multiple splicing factors in defining a specific splicing outcome.3 In all cases, the results from ITC help to design mutations that are tested directly in live organisms, in this case C. elegans, by using fluorescent mini-gene reporters contains mutations identified by the ITC studies.

1. Mackereth et al. (2011) Nature 475, 408-4112. Amrane et al. (2014) Nature Commun. 5, 45953. Mackereth (2014) Worm 3, e991240

Novel developments in the field of calorimetry

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

The underestimated potential of isothermal microcalorimetry for microbiological studies Olivier Braissant1

1University of Basel Hospital, Allschwil, Switzerland

Isothermal microcalorimetry (IMC) is often used in chemistry and physics, but except for isothermal titration calorimetry (ITC), it is not used much by biologists and microbiologists. Still isothermal microcalorimetry is a very sensitive tool to investigate the metabolism of microorganisms such as bacteria, fungi or parasitic worms. Only 10’000 to 100’000 bacterial cells are sufficient to get a signal. In addition, isothermal microcalorimetry can be applied for many purposes including diagnostic, drug susceptibility testing, sterility testing, or investigation focusing on biofilms proliferation on new antimicrobial materials. Isothermal microcalorimetry has very few sample limitations and can be used with solid and opaque samples in which monitoring microbial growth can be difficult, not to say impossible. Here we will present a fews cases where isothermal microcalorimetry has been used in clinical or biomedical context. Studies on the application of isothermal microcalorimetry in the context of urinary tract infection and urosepsis are well suited to demonstrate the applicability of this technique in a clinical context. In addition, recent work on mycobacteria (including M. tuberculosis) will be used to demonstrate the potential of microcalorimetry to work on slow growing pathogens. Finally, application of isothermal microcalorimetry for biofilm research will focus on dental and orthopedic implants. Overall this should give an overview of what can be achieved with isothermal microcalorimeters.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

ITC studies of enzyme activity in solution and suspensions

Peter Westh1

1Roskilde University, Denmark.

Isothermal Titration Calorimetry has a significant, and as of yet poorly exploited potential within chemical kinetics, and a particularly promising application is the characterization of enzyme activity. Thus, ITC provides a direct measure of the rate of any chemical reaction, and does not require any probes, reactant modifications or post-experiment practices. In fact, the typical Michaelis Menten curve will occur directly in the raw data as the experiment progresses. These assets are convenient in conventional enzymology, where the reaction takes place in aqueous solution. In addition, they open up for the use of calorimetric investigations of enzyme activity in colloidal systems. This is important because interfacial processes dominate both in vivo and within industrial applications of enzymes. This presentation will discuss some of the advantages and limitations of ITC within enzyme kinetics, and particularly focus on applications that are difficult to address with conventional methods. The latter includes calorimetric investigations of the hydrolysis of biomass; a process of key importance in upcoming bio-refineries producing sustainable fuels and materials.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Use of isothermal titration calorimetry to investigate and identify novel peptide substrates for prolyl carboxypeptidase. A technology comparison

Anne-Marie Lambeir1

1Laboratory of Medical Biochemistry, University of Antwerp, Belgium, anne-marie.lambeir@uantwerpen.be.

The search for novel peptide substrates for proline specific peptidases has been hampered by the lack of suitable assays that are often too expensive, labour intensive or require the need for derivatization and separation of the reaction products. The purpose of this study was to validate the use of isothermal titration calorimetry to study enzyme kinetics compared to more conventional methods (e.g. MALDI-TOF mass spectrometry, quantitation of released C-terminal Phe using RP-HPLC). This led to the discovery of a novel substrate for prolyl carboxypeptidase, an enzyme involved in body weight regulation.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

KinITC – Kinetic Revolution in Drug Discovery

Pascal Zihlmann,1 Marleen Silbermann,1 Priska Frei,1 Said Rabbani,1 Tobias Mühlathaler,1 Timothy Sharpe,2 Beat Ernst1

1 Institute of Molecular Pharmacy, Pharmazentrum, University of Basel, Klingelbergstrasse 50, CH-4058 Basel, pascal.zihlmann@unibas.ch 2 Biophysics Facility, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4058 Basel

Today drug development still often focuses solely on affinity data (KD) to improve a lead structure. However, affinity values reflect a situation in equilibrium that is often not established in vivo due to pharmacokinetic effects, and KD is therefore not necessarily a reliable parameter for drug efficacy. Binding kinetics seem to be a more accurate indicator for pharmacological activity as they shed light on the rate of formation and lifetime of a drug-target complex. However, studies on structure-kinetic relationships that could guide a kinetic optimization process alongside classical affinity improvement are rare, though highly desirable for medicinal chemistry. The recently introduced analysis tool kinITC that derives kinetic data from ITC may change this situation in the future. Using kinITC, we were able to reanalyze our previously published ITC data on the binding of 29 mannoside ligands to the bacterial adhesin FimH and to derive information on the binding kinetics. We subsequently validated these results by SPR. The analysis of the binding kinetics revealed that although we intended exclusively to optimize interactions between protein and ligand in the bound state (reducing the off rate constant, koff), the improvement in affinity of FimH ligands is also a consequence of a stabilizing the transition state or destabilizing the unbound state (increasing the on rate constant, kon). Congeneric ligand modifications, and structural information from co-crystal structures, allowed us to deduce a strong relationship between a hydrogen bond network and koff, while electrostatic interactions and conformational restrictions upon binding were found to have a stronger impact on kon.

Developments in ITC for applications for challenging areas

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

ITC for Structural Glycosciences tools

Anne Imberty1

1Centre de Recherches sur les Macromolecules Végétales (CERMAV), Grenoble, France, anne.imberty@cermav.cnrs.fr.

A large number of pathogenic microorganisms display receptors for specific recognition and adhesion to the glycoconjugates present on human tissues. In addition to membrane-bound adhesins, soluble lectins are involved in infections caused by the bacteria Pseudomonas aeruginosa and Burkholderia cepacia and by the fungus Aspergillus fumigatus that are responsible for hospital-acquired diseases. ITC has been used in conjunction with structural data for the design of high affinity glycomimetics for anti-infectious therapy. These lectins present several binding sites and are best inhibited by multivalent ligand such as glycosylated dendrimers, liposomes, fullerenes and gold nanoparticles. Characterization of multivalent interactions is a challenge that our group has been assessing with the use of microcalorimetry.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

ITC as a tool for the characterization of self-assembled systems in organic solvents

Laurent BOUTEILLER1

1IPCM, Chimie des Polymères, Université Pierre et Marie Curie - CNRS, France.

Isothermal Titration Calorimetry can help characterizing hydrogen bonded self-assemblies both in water [1] and in organic solvents [2-6]. In a qualitative approach, information can be derived on the mechanism of association (i.e. enthalpy versus entropy driven) [1]. Moreover, it is possible to determine the concentration below which disassembly occurs and thus the phase diagram of a particular system [4]. In a more quantitative approach, it is possible to measure the energetic parameters characterizing a particular system [5,6]. This information can in turn be used to model the concentration and the temperature dependence of all the assemblies present in the solution [6].

1. E. Obert, M. Bellot, L. Bouteiller, F. Andrioletti, C. Lehen-Ferrenbach, F. Boué "Both water and organo-solublesupramolecular polymer stabilized by hydrogen bonding and hydrophobic interactions" J. Am. Chem. Soc., 129,15601-15605, 2007

2. B. Isare, G. Pembouong, F. Boué, L. Bouteiller "Conformational control of hydrogen bonded aromatic bis-ureas"Langmuir, 28, 7535-7541, 2012

3. I. Giannicchi, B. Jouvelet, B. Isare, M. Linares, A. Dalla Cort, L. Bouteiller "Orthohalogen substituentsdramatically enhance hydrogen bonding of aromatic ureas in solution" Chem. Commun., 50, 611-613, 2014

4. M. Dirany, V. Ayzac, B. Isare, M. Raynal, L. Bouteiller "Structural control of bisurea-based supramolecularpolymers: influence of an ester moiety" Langmuir, 31, 11443-11451, 2015

5. A. Arnaud, L. Bouteiller "Isothermal titration calorimetry of supramolecular polymers" Langmuir, 20, 6858-6863,2004

6. M. Bellot, L. Bouteiller "Thermodynamic description of bis-urea self-assembly: competition between twosupramolecular polymers" Langmuir, 24, 14176-14182, 2008

Binding complexity and data quality with ITC

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Any Kd you like

Dr. Joseph Coyle1

1 Astex Pharmaceuticals, Associate Director, Cambridge, United Kingdom.

ITC represents the gold standard for affinity measurements in drug discovery and basic scientific research alike. The availability of lower volume instruments with increased sensitivity coupled with automation and software developments has resulted in an increase in the number of publications containing ITC data. However, many of these papers suffer in terms of experimental design, data quality and most importantly data analysis and interpretation. In this presentation, I will give examples derived from the literature and from in-house data to illustrate the pitfalls users can face and highlight the need for a rigorous and critical approach to the use of ITC.

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

Improved characterization of supramolecular assemblies by non conventional Isothermal Titration Calorimetry experiments

David Landy1, Eléonore Bertaut1

1 COMUE Lille Nord de France, F-59000 Lille, France, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), Université du Littoral-Côte d'Opale, 145 avenue Maurice Schumann, F-59140 Dunkerque, France, landy@univ-littoral.fr

ITC is often described as the method of choice for the quantitative characterization of supramolecular assemblies, as it allows a complete thermodynamic picture of the edifice (binding enthalpy, entropy, free energy and heat capacity variation). In this study, we demonstrate that non conventional ITC strategies could lead to an even more versatile and accurate description. Indeed, while a typical ITC experiment generally implies the titration of one of the two partners (placed in the ITC cell) by the other one (placed in the syringe), it has to be pointed out that alternative experiments may be used and combined. Within this scope, we have demonstrated that a unified model may be used for the treatment of any kind of ITC protocol, for both competitive and non competitive conditions. This model has been associated to a global analysis of combined experiments and to an evaluation of the resulting uncertainties on the thermodynamic parameters (using a priori variance–covariance matrix). We showed that simultaneous treatment of classical titrations and dedicated non conventional experiments may dramatically reduce the uncertainties on each thermodynamic parameter [1], thus allowing the characterization of systems which could not be analyzed by classical experiments. Our approach is illustrated by cyclodextrins and serum albumin complexes (with improvement for both specific and and non-specific interactions). The strategies presented in this work should help to lower the limits of detection and extend the applicability of ITC, thus improving the accuracy and reliability of thermodynamic data.

1. E. Bertaut, D. Landy (2014) Beilstein J. Org. Chem. 10, 2630-2641

2nd MicroCal European Users’ Meeting 26-27 September, 2016 Institut Pasteur Paris, France

10 good reasons to perform ITC experiments

Eric Ennifar1

1Architecture et Réactivité de l’ARN, CNRS/Université de Strasbourg, Strasbourg, France, e.ennifar@ibmc-cnrs.unistra.fr.

ITC is a true in-solution technique and is considered the “gold standard” assay for binding since it directly provides, in one single experiment, the complete thermodynamic binding profile between two molecules, without any labelling requirement. Although ITC is a powerful technique for understanding molecular interactions, it is often only used to provide binding constants (or even misused) and several useful aspects are frequently overlooked by users. Here, several developments from our laboratory will be presented, including the collection of kinetic data in addition to thermodynamic data. Several applications of ITC will be presented and I will show how a good knowledge of ITC can be used to dissect complex molecular interactions.