RESEARCH

Our goal is to use and further develop biophysical approaches to respond to interesting biochemical and biomedical questions and to end up with models explaining function and/or regulation.

Biophysical and NMR data → Structure, Dynamics, Topology → Function

One of many examples investigated by us is the role of membranes in the aggregation of huntingtin. This large protein carries a stretch of polyglutamines that when reaching a critical length led to the development of the hereditary Huntington’s disease. Indeed there is a correlation between the age of onset of the disease and the number of glutamines encoded in the gene, which are thought to be the origin of protein aggregates impairing a number of neurological functions. Once it was realized that the most amino-terminal 17 amino acids directly preceding the polyglutamine stretch play an important role in the spatio-temporal distribution of huntingtin between different cellular organelles and the development of the disease we investigated the structure in micellear environments by multidimensional solution state NMR spectroscopy and its membrane topology by oriented solid state NMR.

       

Figure 1 : Three-dimensional structures of huntingtin 1–17 in DPC micelles. The ribbon representation of the averaged structure (center) shows the alpha-helical conformation from K6 to F17. Hydrophobic side chains are all oriented along one side of huntingtin 1–17, shown for the electrostatic surface potential with unipolar, negatively and positively charged aminoacid residue side chains in white, red, and blue, respectively (right). [illustration taken from this publication]


Notably, using the angular restraints from the solid-state NMR spectra we were able to refine the solution NMR model. Furthermore, a unique solution for the topology and valuable information about the wobbling and rocking motions of the amphipathic domain could be derived from the solid-state NMR data. In this work we also carefully analysed how systematic and statistical errors as well as motions or conformational details affect the resulting tilt and rotational pitch angle analysis.



         

Figure 2 : solid-state NMR spectra (g) of huntingtin 1–17 peptides reconstituted into uniaxially oriented POPC bilayers. Huntingtin 1–17 was labeled with 15N at position Phe17 (a), Phe11 (c), Leu7 (e), or with 2H3 at Ala10 (g). The glass plate normal was aligned parallel to magnetic fiel. Panel g also shows a spectral simulation for a sample mosaicity with Gaussian distribution of 3 and line broadening of 1.5 kHz. [illustration taken from this publication]






Figure 3 : Angular restrictions obtained from solid-state NMR spectra of huntingtin 1–17 reconstituted in oriented POPC bilayers. (a) The possible alignments of the low-energy conformer 3 obtained by solution NMR in the presence of DPC micelles, structure calculation, and refinement are represented by their helical tilt and the rotational pitch angles. The solid black lines represent angular pairs that agree with the experimental 2H quadrupolar splitting obtained from 2H3-Ala-10 (11 +/- 2.5 kHz), the 15N chemical shifts of 15N-Leu-7 (red; 71.5 +/- 2.5 ppm), 15N-Phe-11 (green; 78.5 +/- 3 ppm), and 15N-Phe-17 (blue; 88 +/- 1.7 ppm). The tilt/rotational pitch angular pair is circled where all experimental data agree. (d) Exhibits the pitch angle and tilt angle definitions with some complimentary views to those depicted in (e). (e) Structural details of residues 5–17 are shown when viewed from the side or from the carboy-terminus. The hydrophobic residues are shown in yellow, alanine in gray, serines in green, glutamates in red, and lysines in blue. [illustration taken from this publication]



       
Figure 4 : Structural details of huntingtin 1-17. The labeled sites used for the topological analysis are highlighted by stick and ball models and arrows, the 15N-1H vectors are highlighted in yellow. The conformers 3 and 20 show good agreement with the topology and orientation of the labeled sites, whereas conformer 1 represents a structure that does not match the solid-state NMR topological analysis. [illustration taken from this publication]


Additional experiments were performed: Fluorescence quenching experiments allowed us to deduce the penetration depth; Molecular Dynamics calculation allowed us to analyse in more depth and visualize the membrane associated peptide structure, topology and dynamics; Calcein release experiments to monitor pore forming activities as a function of lipid composition and CD-spectroscopy to quantitatively characterize the reversible association of the huntingtin membrane anchor with bilayers of various composition. The ensemble of data let us propose a model where anchoring the polyglutamine chain to the membrane promotes its aggregation, an hypotheses that has been validated by a number of biophysical approaches used to investigate constructs encompassing the 17-amino acid membrane anchor and polyQ stretches of increasing length.





Figure 5 : Schematic representation of the possible role of Htt17 in the huntingtin aggregation process. The Htt17 domain (blue) anchors huntingtin at membranes concomitant with a structural transition. The resulting alpha-helix (cylinder) is oriented along the membrane surface with the C-terminal phenylalanine (Phe17) and the subsequent polyQ tract (red) facing the cytosol. Membrane-associated Htt17 thereby brings polyQ tracts into the proximity, permitting nucleation that is dependent on the length of the glutamine tract, and the formation of extended à- sheets. High-molecular mass aggregates ultimately result in toxic fibril formation and cell death. In addition, the amphipathic character of the !- helix favors interactions with other protein domains. [illustration taken from this publication]

MAOS NMR and Dynamic Nuclear Polarisation

The MAOSS approach was used to provide proof-of-concept that oriented membranes can be investigated by Dynamic Nuclear Poalrization when a single 13C/15N1H MAS probe was available but no dedicated NMR probe head for oriented samples. The technique allowed us to define the alignment of a transmembrane helica polypeptide and its orientatonal distribution as well as the DNP enhancement factors under higher spinning.





Figure 6 : Oriented membrane samples encompassing the biradical bTbK and a transmembrane peptide carrying a single 15N labeled residue have been prepared on polymer sheets with sample geometries that fit into a 3.2 mm MAS rotor. The proton-decoupled 15N cross-polarization spectra of the peptide were recorded at 100K and characterized by a single line at fast magic angle spinning speeds (≈ 8 kHz). Irradiating these samples with μ-waves resulted in Dynamic Nuclear Polarization and a concomitant 18-fold signal enhancement which considerably shortened the NMR acquisition times. The DNP signal enhancement opens up enhanced possibilities for multidimensional solid-state NMR investigation of oriented membrane polypeptides. [illustration taken from this publication]


In the meantime in a collaborative effort a dedicated flat-coil probe for DNP measurements on oriented membrane samples, a specialized cooling chamber, optimized sample geometries and support materials, biradicals designed for membrane samples and protocols for the best sample preparation have been made available and allowed to record a 2D PISEMA spectrum in less than 2h with only 1/10th of the previously necessary amount of protein.



Figure 7 : Flat-coil probe for DNP measurements on oriented membrane samples. Left side: gas transfer line with ports; Center: complete probe with shielding Dewar; right side: waveguide. [illustration taken from this publication]





Figure 8 : DNP / solid-state PISEMA spectrum of the transmembrane model peptide [15N5]-hΦ19W carrying five consecutive 15N labels reconstituted into uniaxially oriented POPC bilayers. A helical wheel with the 5 positions is shown for comparison.


An ultra-fast MAS probe head is currently under installation on our 750MHz WB spectrometer (September 2016).



Figure 9 : Correlation MAS spectra of the tri-peptide N-Formyl-Met-Leu-Phe.

OPTICAL SPECTROSCOPIES

We use a number of other techniques in the laboratory including light scattering, CD- FTIR- FRET and fluorescence spectroscopies to further define the membrane interactions and functionalities of proteins, peptides and complexes. These applications are found in many of our publications.

Here we only mention a fluorescence quenching approach developed by us recently to detect mesophase arrangements at the surface of lipid bilayers, which shed new light on the active mechanism of antimicrobial peptides and the synergism when some of them are added in combination.




Figure 10 : Model illustrating an end-to-end alignment of in-planar peptides. The grey circles represent the lipid molecules, the boxes represent the peptides and the red circles the critical radius of the NBD label. The picture corresponds to about 10% of peptide / lipid (mol/mol). [illustration taken from this publication]