Rôle de la dynamique conformationnelle dans la fonction protéique

Conformational dynamics in protein function

Methods

NMR

Liquid-Nuclear Magnetic Resonance (NMR) spectroscopy is an atomic-resolution technic amendable to study a range of dynamic timescales that are typical for biological events. Pure or partially purified isotopically labelled proteins can be mixed in media that mimic their physiological environment. Sub-ns dynamics, that is a typical timescale for the conformational sampling of an intrinsically disordered proteins are being probed by 15N spin relaxation measurements. µs to ms transitions, that are of typical timescales for complex association/dissociation, are being probed by chemical exchange experiments (Carr−Purcell−Meiboom−Gill, chemical exchange saturation transfer experiments). Slower dynamics can be probed by real-time NMR spectroscopy. All these methods are being performed on the NMR plateform of the IMM.

Superimposition of different conformational states of the small conditionally disordered protein CP12. Four conformations of the oxydised state CP12 are depicted in colors that range from light to dark blue.
600 NMR spectrometer equiped with a cryoprobe of the IMM NMR plateform. In this spectrometer, the proteins are placed in 5mm NMR tubes (center). On the left, are depicted a biological system that has been characterised by NMR including the conformational ensemble of the intrinsically disordered protein CP12 (red), in isolation or in complex with its partner GAPDH (blue).

Site Directed Spin Labeling and EPR

Comprehending the dynamic nature of protein structures is paramount for unraveling their functions, biological activities, and interactions with other molecules.

Site-Directed Spin Labeling (SDSL) combined with Electron Paramagnetic Resonance (EPR) spectroscopy stands out among magnetic resonance techniques as a valuable tool for probing protein structure and dynamics. This precise and potent method enables the examination of structural alterations and conformational transitions not only in soluble proteins but also in membrane-bound ones. Moreover, its application has revealed significant advantages in elucidating protein dynamics within cellular environments.

SDSL-EPR approach is based on the grafting of a paramagnetic label, usually a nitroxide spin label, at a chosen position on a protein. One advantage of this technique is that we are not limited by the size of the protein or protein-complex, nor by the buffer, pH, environment…

Illustration of the EPR spectral modification due to the folding of a disordered protein induced by its physiological partner.

The EPR spectra shape of the nitroxide is dependent of the mobility of the probe, and subsequently on its local environment. One can then follow the modification of the EPR spectrum at room temperature depending of structural modification.

When a protein contains two paramagnetic sites — whether they are introduced externally or occur naturally — or when one site is present in the protein and the other in its partner molecule, the distances between these sites can be measured. This capability is achieved through pulsed EPR and the DEER (Double Electron Electron Resonance) sequence. Typically, in biological systems, distance measurements span a range from approximately 2 nanometers to 8 nanometers. These measurements can involve pairs of nitroxide labels, or a nitroxide label and a naturally occurring cofactor such as a metal center (e.g., Cu2+, Gd3+, Mn2+), a radical cofactor, an amino acid radical, …..

Illustration of the EPR spectral modification as a function of the mobility of the paramagnetic probe, in the cases of a free label in solution, a probe grafted onto a protein in liquid solution, and in a frozen solution.
Inter-label distance measurements: A) 4-pulse DEER pulse sequence based on the application of two frequencies: the “observed” and the “pump” frequency. B) Echo field sweep (left) of the nitroxide bi-labeled protein with the indication of the chosen frequencies. Background corrected DEER trace (middle) and the resulting distance distribution (right) calculated by DEERAnalysis are shown. C) Echo field sweep (left) of the nitroxide mono-labeled protein reconstituted with Cu(II) in its active site with the indication of the chosen frequencies. Corrected DEER trace (middle) and the resulting distance distribution (right) calculated by DEERAnalysis are shown.

Molecular Dynamics

Molecular dynamics (MD) is a simulation technique in which a protein is modelled as a collection of atoms whose positions and velocities evolve based on classical physics principles and empirical force fields. This approach makes it possible to study protein dynamics, folding, stability and interactions with other molecules at a detailed molecular level.

interconversion between the "closed" and "open" conformations of the hCPR protein.
model of a protein in a water box (left) and example of the evolution of residue flexibility during a molecular dynamics simulation (right).

Partially Ordered Membrane Multilayers

Preparation of partially ordered multilayers of membranes or membrane proteins and determination of g-tensor orientation under different experimental conditions

Membranes can be stacked on Mylar sheets in a two-dimensional order when slowly dried in a controlled atmosphere. The same procedure can be applied to purified membrane proteins that will form layers by hydrophobic interactions between their membrane embedded parts when the detergent concentration is decreased below the CMC.

Mylar sheets coated with partially ordered membranes or membrane proteins are introduced in an EPR tube and placed in the magnetic field in the EPR machine. Spectra recorded at different angles between the magnetic field and the membrane plane will give access to the orientation of the g-tensors of the cofactor under study and thus to the orientation of the protein domain that holds the cofactor. A well-ordered protein domain will give rise to a sharp distribution of g-tensor orientation. Mobility of the protein domain as a function of environmental conditions can thus be evidenced by distinct orientations obtained on samples prepared under different experimental conditions. This allowed us to monitor the 2Fe2S cluster of the Rieske head domain of the bc1 complex in different orientations as a function of the surrounding hydrogen bonding network which is governed by the redox state of the co-factor [Figure and Brugna et al DOI: 10.1073/pnas.030539897]. Proteins are attached to the membrane via flexible linker domains, however, can adopt a multitude of conformations and this will translate to equal  g-tensor amplitudes at all angles in a sample where the cofactors neighboring proteins show sharp orientations. Cytochrome c551m  from Aquifec aeolicus [Baymann et al. DOI: 10.1021/bi011201y) and cytochrome b558/566 from Sulfolobus solfataricus [Schoepp-Cothenet et al.doi: 10.1016/S0014-5793(00)02357-7] are examples for such mobile, membrane anchored proteins studied with this technique.

SAXS and SANS

Small angle x-ray and neutron scattering (SAXS and SANS) are extremely valuable and effective techniques to analyze the conformational dynamics of macromolecules in solution. In particular, they are very well adapted to the study of proteins containing disordered flexible regions.

Furthermore, these techniques are extremely powerful when used in combination with other methods, such as those providing high-resolution information (x-ray crystallography, 3D-modeling, molecular dynamics) or providing local structural information (NMR, EPR, circular dichroism…).

On the other hand, SAXS is also particularly well suited for providing experimental constraints allowing to refine calculated 3D-models (AlphaFold, …) especially again when the 3D-structures are poorly predicted because of protein disorder or flexibility, or the lack of homologous sequence.

SAXS data contain all the 3D information on the various conformations adopted by a macromolecule in solution. Indeed SAXS not only provides the global dimensions and shape of the protein (Radius of gyration, maximum dimension, distribution of intramolecular distances, average shape of the macromolecule), but it can also discriminate atomic models compared to the experimental structure in solution. In particular, by calculating the SAXS profile from atomic coordinates and comparing it with experimental SAXS data, it becomes possible to select an ensemble of conformations that collectively contribute to the observed scattering spectrum, thus accounting for the conformational flexibility of the protein.

Distribution of conformation of oxidized CP12 in solution : the SAXS data (right, black curve) are very well fitted (right, red curve) by a distribution of conformation (left, black histogram) corresponding to the sum (left, grey curve) of two Gaussian distributions of conformations: 60% of the ensemble of conformations where the N-terminus of CP12 adopts a helical hairpin conformation with other disordered regions (blue curve), and 40% of the ensemble of conformations where the N-terminus of CP12 is unfolded (green curve), that are in equilibrium in solution. Adapted from Launay et al. 2018, J. Mol. Biol.