Experimental NMR dataset related to the publication in Magnetic Resonance 2021 entitled: "Fluorine NMR study of proline-rich sequences using fluoroprolines" by Davy Sinnaeve, Abir Ben Bouzayene, Emile Ottoy, Gert-Jan Hofman, Eva Erdmann, Bruno Linclau, Ilya Kuprov, José C. Martins, Vladimir Torbeev and Bruno Kieffer Abstract. Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering – they introduce conformational and dynamical biases – but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two postions in distinct polyproline segments. By looking at the effects of swapping these (4R)- and (4S)-4-fluoroprolines within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and exploit the latter in elucidating binding kinetics to the SH3 domain CONTENT OF THE REDEPOSITORY Peptide assignment To assign MpRS and MpSR peptides we recorded at 298 K the following experiments: H-13C HSQC. Recorded at 700MHz. The corresponding data are in the following datasets: MpRS_HSQC and MpSR_HSQC H-1H TOCSY. Recorded at 600MHz. The corresponding data are in the following datasets: MpRS_TOCSY and MpSR_TOCSY H-13C HSQC-NOESY. Recorded at 700MHz. The corresponding data are in the following datasets: MpRS_HSQC_NOESY and MpSR_HSQC_NOESY H-1H NOESY. Recorded at 600MHz. The corresponding data are in the following datasets: MpRS_NOESY and MpSR_NOESY Homonuclear scalar coupling constants Homonuclear 3JH-H scalar couplings were measured using SERF experiments on a 700MHz spectrometer at 298 K. The corresponding data can be examined in the followed datasets: MpRS_SERF and MpSR_SERF for both MpRS and MpSR peptides, respectively. 1D 19F spectra 1D 19F spectra of MpRS and MpSR peptides were recorded on a 600MHz spectrometer at 298 K, with samples dissolved in D2O. 3mm tubes were used. The data are stored in the following datasets: MpRS_1D_F and MpSR_1D_F for both MpRS and MpSR peptides respectively. 19F Relaxation Longitudinal and transverse relaxation rates of fluorine were measured for both MpRS and MpSR peptides. Standard inversion recovery, spin-echo and CPMG experiments were collected for both MpSR and MpRS peptides on a 600MHz at 298 K. The corresponding data may be examined in these datasets: For MpRS: MpRS_R1, MpRS_R2_spin_echo, MpRS_R2_CPMG For MpSR: MpSR_R1, MpSR_R2_spin_echo, MpSR_R2_CPMG 1HG-19F heteronuclear-nOes were measured using nOe buildup experiments recorded on a 600MHz spectrometer at 298 K. The experimental data of both peptides are in the following datasets: MpRS_HGF_NOE and MpSR_HGF_NOE Titration experiments with Vinexin SH3.3 domain Titration experiments were performed on a 600MHz spectrometer at 298K using 3 mm tubes. The respective volumes of peptide and SH3 protein are given in the title file. The 1D 19F spectra of MpRS and MpSR peptides are stored in titrMpRS_1D_F and titrMpSR_1D_F, respectively. The 1H-15N HSQC spectra are stored in titrMpRS_HSQC and titrMpSR_HSQC datasets. Stock solutions were: SH3: 314 µM MpRS S0: 5.67 mM MpSR S0: 5.1 mM S1 stocks resulted from S0 by a factor 2 dilution.