Unit: Biophysics of Macromolecules and their Interactions (Platform)
Director: Patrick ENGLAND
The Centre of Biophysics of Macromolecules and their Interactions (french acronym: PFBMI) is a resource facility including an important number of methodologies allowing the study of the physico-chemical properties of biological macromolecules and their complexes.
The 12 instruments managed by this facility are open to all investigators present on the campus of the Pasteur Institute, as well as to the scientific community in general.
An e-mail address, firstname.lastname@example.org, is available for any person wishing to submit a project to the PFBMI.
The Centre of Biophysics of Macromolecules and their Interactions regroups a set of technologies which is unique in France. These allow the characterisation of both intrinsic properties of macromolecules (stability, folding, self-association, ) and those of the interactions in which they are involved (thermodynamic and kinetic parameters, stoichiometry, ).
* Surface plasmon resonance (scientific manager : Patrick ENGLAND ; technician : Sylviane HOOS).
The Biacore 2000 instrument (Biacore ; http://www.biacore.com) allows the real-time analysis of molecular interactions. To that avail, one of the partners of the interaction is immobilised, covalently or non-covalently, and the other partner is brought in contact with this surface by a continuous flow of buffer. This technique is especially well adapted to the measurement of the rates of association (kon) and dissociation (koff) of biological macromolecules. The instrument is entirely automated, which makes it easier to accomplish highly reproducible series of experiments.
* Microcalorimetry (scientific manager : Francis SCHAEFFER ; technician : Sylviane HOOS).
Microcalorimetry allows the direct, thorough and accurate thermodynamic characterisation of interactions between molecules in solution and of biochemical reactions in general. Two types of calorimeters (Microcal ; http://www.microcalorimetry.com/) are available at the PFBMI, depending on the focus of the study that one wishes to perform:
Isothermal titration calorimetry (MCS-ITC and VP-ITC models): principally dedicated to the study of molecular interactions. By measuring the reaction heat at constant temperature, ITC allows the direct determination of the stoichimetry (n), the equilibrium constant (Ka), and the variations of enthalpy (ÆH) and entropy (ÆS) induced by an association reaction. By performing titrations at several temperatures, it is possible to measure the variation of heat capacity (ÆCp) related to the formation of a molecular complex.
Differential scanning calorimetry (VP-DSC model): principally dedicated to the study of the thermal stability of macromolecules and their complexes. By measuring the variation of heat capacity (ÆCp) as a function of temperature, DSC allows to determine directly the variations of enthalpy (ÆH) and entropy (ÆS) and the melting temperature (Tm) related to each structural transition. The Pressure Perturbation Calorimetry accessory (PPC), the first of its type in France, allows the determination of the volumetric properties of macromolecules, such as their coefficient of thermal expansion.
* Circular dichroism (scientific manager : Alain CHAFFOTTE).
The PFBMI has an Aviv 215 circular dichroism instrument (http://www.avivbiomedical.com), including total fluorescence and titration accessories. It also has an older spectropolarimeter (Jobin-Yvon CD6) equipped with a "stopped-flow" accessory (Bio-Logic).
The study of macromolecules (and particularly proteins) by circular dichroism allows the analysis of the secondary and tertiary structures. For proteins, the measure of circular dichroism in the far UV (180-260 nm) allows to determine the secondary structure content, while the near UV signals (250-330 nm) give information about the tertiary folding.
The thermal stability of the macromolecular folding can be determined by applying a temperature gradient to the sample. In a similar fashion, it is possible to quantify the conformational stability of a molecule at constant temperature as well as the structural changes induced by the formation of complexes by performing a titration, respectively by a denaturing agent or a ligand. Finally, kinetic studies can be performed with a rapid mixing device ("stopped-flow").
* Infrared spectroscopy (scientific manager : Alain CHAFFOTTE).
Fourier transform infrared spectroscopy (FTIR) allows the study of the secondary structure of macromolecules, by analysing the absorption frequencies of characteristic chemical moieties (notably the peptide bond in the case of proteins). This technique yields data that complement those provided by circular dichroism, and is especially well suited to the study of the secondary structure of proteins with a high content of ß-sheet structures. The instrument available at the PFBMI (MB104 ; ABB Bomem ; http://www.abb.com/analytical) has been equipped in 2004 with a DuraSamplIR attenuated total reflectance accessory (SensIR; http://www.sensir.com/newsensir/in_lab/DuraSamplIRII.html).
* Analytical ultracentrifugation (scientific manager : Thierry ROSE).
The hydrodynamic behaviour and the sedimentation coefficient of a macromolecule are related to its volume, shape, density and mass. Analytical ultracentrifugation allows a direct measurement of the mass of a macromolecule in solution: if it is an oligomer or a non-covalent complex, the number of chains that make up the native molecule or the stoichiometry of the complex can therefore be determined. Information can also be inferred about the conformation of macromolecules and the equilibrium constant of their assemblies, as well as about the homogeneity of samples. The PFBMI has acquired in 2004 a ProteomeLab XL-I analytical ultracentrifuge equipped with a double detection system (UV/visible and Rayleigh interference optics), which adds to an already available Optima XL-A ultracentrifuge which has been recently upgraded (Beckman-Coulter ; http://www.beckman.com/products/splashpage/xla/default.asp).
* Fluorescence spectroscopy (scientific manager : Fabrice AGOU).
Fluorescence spectroscopy is a versatile technique that allows the analysis, both at equilibrium or in real time, of the variations of the environment of fluorescent probes, whether these are intrinsic (notably tryptophans in the case of proteins) or extrinsic (synthetic fluorophores). Its applications range from the study of the conformational stability of macromolecules, to the characterisation of molecular interactions and the quantification of enzymatic activities. The instruments available at the PFBMI allow the measurement of both fluorescence intensity and fluorescence polarisation (or anisotropy).
The PFBMI has both a high-sensitivity standard fluorimeter (Quantamaster C60 ; Photon Technology International ; http://www.pti-nj.com/quantamaster.html) and a stopped-flow instrument (KinetAsyst SF61-DX2 ; Hi-Tech ; http://www.hi-techsci.com/), which allows the study of fast reactions with a resolution better than 0.1 s.
* Dynamic light scattering (scientific manager: Patrick ENGLAND)
The study of particles in solution by dynamic light scattering allows the quantification of their diffusion rate and of their hydrodynamic radius (which is related to their mass and their shape). The instrument available at the Pasteur Institute (DynaPro MS800 ; http://www.wyatt.com/solutions/hardware/DynaProTitan.cfm) is especially well suited for the study of the homogeneity of macromolecular preparations, and allows the detection of high molecular weight aggregates with a very good sensitivity.
The PFBMI has participated since 2002 in more than 70 projects, involving 20 Research Units of the Pasteur Institute, from 8 different Scientific Departments, and 15 laboratories from other institutions. The activity has been distributed between routine experiments and more thorough collaborative work.
The PFBMI has notably been involved in the Federative Horizontal Program (GPH) on Tuberculosis.
Many projects have required the use of two or more methodologies available at the PFBMI.
14 peer-reviewed scientific publications co-authored by scientific supervisors of the PFBMI have resulted from collaborations within or beyond the Pasteur Institute.
Figure 1 : Study of the kinetics of interaction between macromolecules by surface plasmon resonance (Biacore).
Figure 2 : Aviv215 Circular Dichroism instrument.
Figure 3 : ProteomeLab XL-I analytical ultracentrifuge.
Keywords: physico-chemistry, spectroscopy, thermodynamics, kinetics, proteins, saccharides, nucleic acids