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Solid-State NMR Study Reveals Collagen I Structural Modifications

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Solid-State NMR Study Reveals Collagen I Structural Modifications Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis Paulo de Sa Peixoto, Guillaume Laurent, Thierry Azais, Gervaise Mosser To cite this version: Paulo de Sa Peixoto, Guillaume Laurent, Thierry Azais, Gervaise Mosser. Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2013, 288 (11), pp.7528-7535. 10.1074/jbc.M112.390146. hal-01461409 HAL Id: hal-01461409 https://hal.archives-ouvertes.fr/hal-01461409 Submitted on 2 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Solid-state NMR study reveals Collagen I structural modifications of amino acid side chains upon fibrillogenesis. Paulo De Sa Peixoto, Guillaume Laurent, Thierry Azaïs, Gervaise Mosser. Laboratoire de Chimie de la Matière Condensée de Paris, UMR7574-UPMC/CNRS/Collège de France, UPMC, 4 place Jussieu, 75005 PARIS, France. Running Title : fibrillogenesis of Collagen I studied by solid-state NMR To whom correspondence should be addressed: Gervaise Mosser, Laboratoire de Chimie de la Matière Condensée de Paris, UMR7574 UPMC/CNRS/Collège de France, 75005 Paris, France. E-mail: [email protected]. Tel: +33 144276553, Fax: +33 144276539. Key-Words: Collagen I; solid-state NMR; fibrillogenesis; random coiled side-chain chemical shift; imino acids Background : Collagen fibrillogenesis is a amino acids residues. Moreover, based on a fundamental process both in vivo and in 13C quantitative spectrum we estimate the vitro. percentage of residues implicated in each Results : Fibrillogenesis increases the equilibrium. Our data indicate that fibril heterogeneity of conformations of side formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. chains amino acids, impacting 40% of Finally, we discuss the implication of these iminoacids. structural data and propose a model in which Conclusion : A temperature-induced- the attractive force of fibrillogenesis comes attractive force component comes from from a structural reorganization of 10% to significant amount of the collagen amino 15% of the amino acids. These results allow acids. us to further understand collagen's self- Significance: Brings new perspectives to assembling process and fibrillar structure. studies of collagen assembly and ____________________________________ interactions with other matrix molecules. Collagen I, the most abundant protein in human body, is a very long (300 nm) and thin (1.5 nm) SUMMARY protein. It is formed by three left-handed In vivo, collagen I, the major structural polypeptide chains super-coiled into a right- protein in human body, is found assembled handed helix. These polypeptide chains, into fibrils. In the present work, we study a composed of about 1040 amino acids each (1), high concentrated collagen sample in its are formed almost exclusively by repeats of soluble, fibrillar and denatured states using 1 13 three amino acid units -(Gly-X-Y) - where Gly one and two dimensional { H}- C solid-state n 13 is a glycine and X and Y can be any other amino NMR spectroscopy. We interpret C acid but often, in mammals, X is a proline and Y chemical shifts variations in terms of dihedral is a hydroxyproline (2). The collagen molecule angle conformation changes. Our data show has propensity to self-associate into fibrils that that fibrillogenesis increases the side chain further organize into hierarchical architectures and backbone structural complexity. both in vivo and in vitro (3, 4). Within those Nevertheless, only three to five rotameric units, collagen molecules are arranged equilibria are found for each amino acid longitudinally and laterally so as forming a residue indicating a relatively low structural crystal-like period called “D period” of 67 nm heterogeneity of collagen upon fibrillogenesis. evidenced both by X-ray diffraction (XRD) and Using side chain statistical data we calculate electron microscopy studies (5,6). So far, the equilibrium constants for a great number of 1 highest resolution obtained on the whole molecule by XRD is 5-11 Å (lateral and axial EXPERIMENTAL PROCEDURES resolution respectively) (5). High-resolution Sample purification and fibrillogenesis local structural data were obtained on several Collagen was prepared as already described (6). crystals of collagen-like peptide. However, due Collagen concentration was assessed by to the limited number of these crystals (~20 hydroxyproline titration (16) and purity was resolved structures currently in the PDB) data determined by SDS-PAGE electrophoresis. collected are far from representing the great Collagen stock solution (500 mM CH3COOH, diversity of -(Gly-X-Y)n- sequences present in pH=2.5) was dialyzed against HCl (3 mM, collagen I (7). 1H NMR has only shown pH=2.5) and concentrated to 410 mg.mL-1 by a moderate potential to solve collagen-like peptide centrifugation-filtration process using PES tubes structure because of many overlaps of the of 30 KDa cut-off (3000 g at 10°C, variable resonances (8). Moreover, up to now 13C NMR angle centrifuge EPENDORF 5702R™). The pH experiments did not display a sufficient spectral was further increased by exposing the acidic resolution to allow a clear distinction among the sample to ammonia vapors (30 w %) at 20°C for different amino acid carbon resonances (9, 10). 7 minutes. 2D solid state NMR spectra were NMR studies using specific residue labeling recorded 6 hours after the sample exposition to have given valuable information on dynamics ammonia and acquired during 6 hours. The (11, 12, 13). In order to deduce the backbone stability of the sample was checked by recording conformation, Saito H. et al have compared 13C 1D 13C spectra prior and after each 2D 1H-13C chemical shifts of fibrillar collagen with those experiments. The spectra were similar for each obtained on small triple helical peptides (9). condition. Finally, the “basic-pH” sample was Torchia et al. have compared native and centrifuged-filtrated in order to collect enough denatured resonances of collagen fragment CB2 liquid (~150 µl) to measure the pH with a pH (14). More recently, Zhu et al. have studied paper. The resulting pH was 8.5 ± 0.5. structural modifications of bovine cortical bone Denaturation of the sample was obtained by induced by dehydration (10). Even though all exposing the sample to a high power 1H these studies reveal important structural decoupling (ν(1H) = 60 kHz) for 16 hours. characteristics of collagen, many 13C chemical shifts were “hidden” due to a poor spectral NMR parameters resolution. Solid-state NMR analyses were performed on a In the present work, we have investigated, using Bruker Avance III 500 spectrometer at 11.7 T solid-state NMR, structural changes in collagen operating at 500.21 MHz for 1H and 125.79 at high concentrations in four different regimes: MHz for 13C. A Bruker Magic Angle Spinning three native conditions defined as (i) soluble at (MAS) double resonance probe was used with 7 acidic pH (pH 2.5; T = 20°C), (ii) and (iii) mm rotors spun at a frequency νMAS = 2.5 kHz fibrillar at basic pH (pH 8.5; T = 20°C and T = A BCU-X unit was used to regulate the 30°C), and a denatured condition (iv) (pH 8.5; T temperature between 20°C and 35°C. For all 1 13 = 35°C). This last sample was used as a experiments, t90( H) and t90( C) were 11 and 7 reference. We have observed spectrally resolved µs respectively. Relaxation delays of 1 and 3 s 1 13 13 1D and 2D { H}- C MAS spectra ( C typical for 1H and 13C respectively were sufficiently line width from 20 to 40 Hz). We have identified long to record quantitative spectra. 512 scans 13 side chains and backbone C chemical shifts were acquired for 13C single pulse-experiment, modifications upon fribillogenesis passing from 48 scans for the 2D {1H}-13C INEPT MAS acidic to basic pH. We have shown that one experiment. For all 13C spectra a low power given residue can adopt different conformations proton decoupling (4.5 kHz) was applied during along the molecule. We have interpreted our acquisition. The INEPT evolution delay (1.7 ms) 1 data in terms of side chain conformational was matched to an averaged JCH value of 145 modifications using statistical data from ref (15). Hz and the refocusing delay (1.1ms) was We have also quantified those structural matched in order to get the CH, CH2 and CH3 13 modifications by comparing the C chemical resonances similarly phased. 256 t1 transients shifts of denatured collagen to side chain were collected for 2D {1H}-13C INEPT spectra. structural library data. 1H and 13C chemical shifts were referenced (δ = 0 ppm) to TMS. No 13C signal was detected through a cross polarization (CP) MAS 2 experiment (contact time of 10ms, 440 scans and have been fitted (SD always inferior to 6% at pH a 1H decoupling of 40kHz). See SI section figure 8.5). 2. Assignment and fit RESULTS 1H and 13C chemical shifts assignments were Dense collagen I matrices were synthesized based on 2D {1H}-13C INEPT MAS spectra (Fig. through a two-step procedure making use of the 1, Fig. SI-1), and on previous works on a lyotropic properties of collagen molecules at collagen-like peptide (8) and native collagen I acidic pH and self-assembling properties at (9, 10, 17).
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