High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations
Abstract
:1. Introduction
2. Results and Discussion
2.1. Chemical Shifts Assignment of RGDechi1-14 and ψRGDechi
2.2. Chemical Shifts Analysis of RGDechi1-14 and ψRGDechi
2.3. 3JHNHα Coupling Constants, HN Temperature Coefficients and NOEs Evaluation
2.4. Structural Comparison of RGDechi1-14 and ψRGDechi versus RGDechi
2.5. RGDechi1-14 and ψRGDechi Backbone Motions
2.6. Conformational Ensemble of RGDechi1-14 and ψRGDechi Peptides
2.7. Elucidation on the Recognition Mechanism of αvβ3 Integrin by ψRGDechi
3. Materials and Methods
3.1. Peptides Preparation
3.2. NMR Experiments
- (i)
- The 2D [1H-1H] TOCSY (total correlation spectroscopy) [55], 2D [1H-1H] NOESY (nuclear overhauser effect spectroscopy) [56], and 2D [1H-1H] ROESY (rotating frame overhauser effect spectroscopy) [57] data were acquired using 32 scans per t1 increment, a spectral width (SW) of 6001.30 Hz along both t1 and t2, 2048 × 256 complex points in t2 and t1, and a relaxation delay of 3.0 s. In all NMR experiments reported above, the water suppression was achieved using the Watergate pulse sequence with the application of gradients using double echo. The 2D [1H-1H] TOCSY experiments were acquired by homonuclear Hartman–Hahn transfer by a MILEV17 mixing sequence with a mixing time (tm) of 70 ms and 10 KHz spin-lock field strength; The 2D [1H-1H] NOESY spectra were recorded using a mixing time of 250 ms and 2D [1H-1H] ROESY were carried out with a cw spin-lock field strength of 4 kHz, a mixing time of 250 ms, and a relaxation delay of 5.0 s. All homonuclear 2D experiments were apodized with a square cosine window function and zero fill to a matrix of size 4096 × 1024 before Fourier transform and baseline correction.
- (ii)
- Heteronuclear natural-abundance 2D [1H–15N] HSQC (heteronuclear single quantum coherence) experiments were performed with 880 scans per t1 increment, SW of 1581.32 and 6001.30 Hz along t1 and t2, respectively, 2048 × 128 complex points in t2 and t1, respectively, and a 1.0 s relaxation delay. In particular, the 2D [1H–15N] HSQC pulse sequence was optimized as previously reported [16] using a Bruker standard gradient echo–antiecho pulse program [58]. Two-dimensional [1H–15N] HSQC spectra were apodized with a square cosine window function and a zero filling to a matrix of size a 2048 × 128 before Fourier transform and baseline correction. Natural-abundance 2D [1H–13C] HSQC spectra were acquired with 880 scans per t1 increment, SW of 5130.94 Hz along t1 and 6001.30 Hz along t2, 2048 × 200 complex points in t2 and t1, respectively, and a relaxation delay 1.0 s. The 2D constant time (CT) [1H–13C] HSQC pulse sequence was optimized as reported is a previous publication [16]. Two-dimensional CT [1H–13C] HSQC were acquired using a heteronuclear coupling constant (JXH) of 145 Hz, CT period of 26.6 ms, and shaped pulses for all 180° pulses on f2 channel with decoupling during f1 acquisition. Two-dimensional CT [1H–13C] HSQC spectra were apodized with a square cosine windows function and zero fill to a final matrix of 4096 × 4096 before Fourier transform and baseline correction.
- (i)
- Two-dimensional T2-filter [1H–15N] HSQC experiments were carried out using 880 scans per t1 increment, SW of 1581.32 Hz along t1 and 6001.30 Hz along t2, 2048 × 128 complex points in t2 and t1, respectively, and 1.0 s relaxation delay. In particular, for each peptide, the dynamics characterization was performed by two 2D T2-filter [1H–15N] HSQC spectra acquired using different relaxation compensated CPMG (Carr–Purcell–Meiboom–Gill) sequence periods (125 and 250 ms). Notably, in this [1H–15N] HSQC-based experiment only the 1H-15N cross-peaks of the residues for which the 15N transversal relaxation time (T2) was longer than the applied filter were observable. The pulse sequence was optimized as previously reported [16]. Two-dimensional T2-filter [1H–15N] HSQC spectra were apodized with a square cosine window function and zero filling to a matrix of size 4096 × 1024 before Fourier transform and baseline correction.
3.3. Chemical Shifts Analysis
3.4. Amide Temperature Coefficients and 3JHNHα Scalar Couplings Analysis
3.5. Molecular Dynamics Simulations
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Acconcia, C.; Paladino, A.; Valle, M.d.; Farina, B.; Del Gatto, A.; Gaetano, S.D.; Capasso, D.; Gentile, M.T.; Malgieri, G.; Isernia, C.; et al. High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations. Int. J. Mol. Sci. 2022, 23, 11039. https://doi.org/10.3390/ijms231911039
Acconcia C, Paladino A, Valle Md, Farina B, Del Gatto A, Gaetano SD, Capasso D, Gentile MT, Malgieri G, Isernia C, et al. High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations. International Journal of Molecular Sciences. 2022; 23(19):11039. https://doi.org/10.3390/ijms231911039
Chicago/Turabian StyleAcconcia, Clementina, Antonella Paladino, Maria della Valle, Biancamaria Farina, Annarita Del Gatto, Sonia Di Gaetano, Domenica Capasso, Maria Teresa Gentile, Gaetano Malgieri, Carla Isernia, and et al. 2022. "High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations" International Journal of Molecular Sciences 23, no. 19: 11039. https://doi.org/10.3390/ijms231911039