Study of the energy landscape of membrane proteins:
application to seven trans-membrane α-helix receptors

Laurent J. Catoire (CNRS Researcher),
Marina Casiraghi (Post-doc labex Dynamo),
Elodie Point-Bonnet (CNRS technician)

LaurentCatoire Laurent J. Catoire

To fully characterize a biomolecule, and so to decipher its function, usually the solely atomic description of a low energy, i.e. predominantly populated and long-lived, conformational state is not enough [1]. Indeed, countless biological functions are closely linked to changes in spatial and temporal location of groups of atoms in biomolecules. In the formalism of an energy landscape, to characterized a biomolecule at a molecular level, it is essential to: i) census the number of conformations co-existing or existing along the functional pathway; ii) to get a 3D description of these structures; iii) to determine the relative probabilities of existence of these different sub-states (thermodynamics); iv) to detect and characterize chemical exchanges, i.e. the kinetic barriers separating these sub-states and associated energies of activation (kinetics).

In the context of investigating the energy landscape of large proteins or protein complexes like membrane proteins, NMR spectroscopy represents a powerful technique when associated to appropriate isotope-labeling schemes (e.g. [2]), methodology [3, 4] and technology (high magnetic fields, cryogenic probes, ...). This spectroscopy can determine at physiological temperatures the conformational ensemble, including conformations that are lowly populated (until 0.5%) and transiently formed (ms), and can characterize in details the chemical exchanges between the different substates detected. This includes determinations of constant rates and populations involved in the different kinetic barriers but also the determination of activation energies and enthalpic and entropic contributions to these exchanges.

In the lab, we focus on the energy landscape of membranes proteins, with a recent example that concerns a G Protein-Coupled Receptor (GPCR) in a lipid bilayer (cf. Figure herein) [5]. The conformational dynamics of GPCRs is central to their signaling plasticity and allosteric regulation but still poorly delineated. In particular, the impact of the membrane environment is barely understood, although it has been repeatedly demonstrated that it plays a central role in membrane protein structure and dynamics. That type of study also requires developing new tools or protocols, like the use of innovative molecules or supramolecular structures to fold and maintain membrane proteins stable and active in vitro, like amphipols [6] and nanodiscs [7].

Conformational landscape of unliganded GPCR BLT2 in nanodiscs.
2D 1H-13C SOFAST-methyl-HMQC/TROSY spectrum acquired with [U-2H,12C]Ile-[δ1-13CH3], [U-2H,12C]Met-[ε-13CH3] BLT2 in low-sterol content nanodiscs in the unliganded state. (a) Global view of the [ε-13CH3]-Met and [δ1-13CH3]-Ile229 regions (boxed in orange); the additional weak peaks correspond to residual lipid signals. (b and c) From panel a, close-ups of the ε-13CH3-Met region and [δ1-13CH3]-Ile229 region, respectively. In panel b, the red spectrum corresponds to that of a mutant receptor that contains the transmembrane Met residues 105 and 197. The peak labeled V in parentheses in panel c is not included in the present analysis of the BLT2 conformational ensemble. Identical spectra were obtained from measurements with two independent samples (from ref. [5]).

Bibliography (in blue from the lab):
  1. Sekhar & Kay (2013). NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc Natl Acad Sci USA, 110, 12867-74 (Pubmed)
  2. Plevin & Boisbouvier (2012). Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins. In Recent Developments in Biomolecular NMR, (Ed. Clore and Potts) .
  3. Pervushin et al. (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA, 94, 12366-71 (Pubmed)
  4. Rosenzweig & Kay (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem, 83, 291-315 (Pubmed)
  5. Casiraghi et al. (2016) Functional modulation of a GPCR conformational landscape in a lipid bilayer. J Am Chem Soc, 138, 11170-5 (Pubmed)
  6. Popot (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem, 79, 737-7 (Pubmed) ;
    Popot et al. (2011) Amphipols from A to Z. Annu Rev Biophys, 40, 379-408 (Pubmed)
  7. Ritchie et al. (2009). Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs. In Methods in Enzymology, (Elsevier), pp. 211-231 (Pubmed)

Last update: 17/08/22

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