Figure 2.

NMR analysis demonstrates that the conserved motifs BRCA2_48–284, BRCA2_250–500, BRCA2_1093–1158, BRCA2_2213–2342 are disordered in solution. (A) Localization of the four fragments in the BRCA2 sequence. (B) 2D NMR ¹H-¹⁵N Heteronuclear Single Quantum Coherence (HSQC) spectra of the four fragments. These spectra were recorded on ¹⁵N-labeled BRCA2_48–284 at 200 μM in HEPES 50 mM, NaCl 75 mM, EDTA 1 mM, DTT 5 mM, pH 7.0 (green, [53]), ¹⁵N-labeled BRCA2_250–500 at 200 μM in phosphate 20 mM, NaCl 250 mM, pH 7.0 (red, 600 MHz CEA Saclay), ¹⁵N-labeled BRCA2_1093–1158 at 200 μM in 50 mM HEPES, 50 mM NaCl, 1 mM EDTA pH 7.0 (blue, 700 MHz CEA Saclay) and ¹⁵N-labeled BRCA2_2213–2342 500 µM, in HEPES 50 mM, NaCl 50 mM, EDTA 1 mM, pH 7.0 (pink, [54]). Cysteines of BRCA2₄₈–₂₈₄ (C132A, C138A, C148A, C161A) and BRCA2_250–500 (C279A, C311S, C315A, C341A, C393S, C419S, C480S) were mutated in order to favor their solubility. Grey backgrounds define the ¹H frequencies typical for disordered residues. Localization of most ¹H-¹⁵N peaks in the grey regions highlights the disorder propensity of these BRCA2 fragments; (C) Secondary structure propensity of three of these fragments. Propensities to form α-helices or β-strands were calculated for BRCA2_48–284 (green), BRCA2_1093–1158 (blue) and BRCA2_2213–2342 (pink) from their ¹HN, ¹⁵NH, ¹³CO, ¹³Cα and ¹³Cβ NMR resonance frequencies using the ncSPC server [56]. Bold black lines underline the presence of transient secondary structure with the indicated percentage. BRCA2_48–284 contains two shorts transient α-helices formed by residues 100 to 110 and 255 to 260, BRCA2_1093–1158 exhibits one transient α-helix between residues 1122 and 1128 and BRCA2_2213–2342 shows one transient α-helix between residues 2292 and 2303.