Unit: Biophysics of Macromolecules and their Interactions
Director: Patrick ENGLAND
The Centre for Biophysics of Macromolecules and their Interactions 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 10 instruments managed by this core facility are open to all investigators present on the campus of the Pasteur Institute, as well as to the scientific community in general.
Created in july 2002, the Centre for Biophysics of Macromolecules and their Interactions (french acronym: PFBMI) regroups a set of physico-chemical technologies which is unique in France. These allow the characterisation of either 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 (knn) 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 model): 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 (?H) and entropy (?S) induced by the association reaction. By performing titrations at several temperatures, it is possible to measure the variation of heat capacity (?Cp) related to the formation of the 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 (?Cp) as a function of temperature, DSC allows to determine directly the variations of enthalpy (?H) and entropy (?S) and the melting temperature (Tm) related to each structural transition.
* Circular dichroism (scientific manager : Alain CHAFFOTTE ; technician : Roland NAGEOTTE).
The PFBMI has a spectropolarimeter equipped with a "stopped-flow" accessory (model CD6 ; Jobin-Yvon Horiba), and has acquired in 2003 a new generation circular dichroism instrument: the Aviv 215, manufactured by Proterion (http://www.proterion.com/avivcd.html).
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) will shortly be equipped with an attenuated total reflectance (ATR) accessory.
* Analytical ultracentrifugation (scientific manager : Michel GOLDBERG ; technician : Roland NAGEOTTE).
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. At present, the PFBMI has an Optima XL-A ultracentrifuge (Beckman-Coulter ; http://www.beckman.com/products/splashpage/proteomelab/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 classical 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 managers: Patrick ENGLAND and Ahmed HAOUZ)
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 (DynaProMS800 ; Proterion ; http://www.proterion.com/ps_dynapro.html) 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 in 2003 in more than 40 projects, involving about 15 Research Units of the Pasteur Institute and 10 laboratories from other institutions. The activity has been evenly distributed between routine services and more thorough collaborative work. Many projects have required the use of two or more methodologies available at the PFBMI.
Fig. 1 : Biacore 2000 instrument : study of the kinetics of interaction between macromolecules..
Fig. 2 : Isothermal titration calorimetry (ITC) : association of an enzyme with a substrate analogue.
Fig. 3 : Far UV circular dichroism spectrum of the catalase/peroxidase KatG of Mycobacterium tuberculosis
Keywords: physico-chemistry, spectroscopy, thermodynamics, kinetics, proteins, saccharides, nucleic acids