NMR insights into the core of GED assembly by H/D exchange coupled with DMSO dissociation and analysis of the denatured state.

摘要: Abstract GTPase effector domain (GED) of dynamin forms megadalton-sized assembly in vitro, rendering its structural characterization highly challenging. To probe the core GED assembly, we performed H/D exchange native state and analyzed residual amides following dissociation by dimethyl sulfoxide (DMSO). The data indicated a hierarchy solvent exposure: Ser2–Glu13, Glu23–Phe32, Asp37–Gln43, Val51–Met55, Lys60–Asp64 followed remaining segments. This reflects chain packing assembly. segment Leu65–Pro138 C-terminal half is largely interior core, while N-terminal Asp37–Asp64 traverses into out core. Next, characterized motional behavior DMSO-denatured state. stretches Gly9–Lys18, Asp37–Arg42, Lys68–Met74, Ser136–Thr137 were seen to display alternate conformations slow exchange. In major population, both α β propensities along polypeptide chain. Spectral density analysis 15N R1, R2, 1H-15N nuclear Overhauser effect collected at 600 800 MHz suggested presence four domains motions, namely, A (Leu40–Tyr91), A′ (Leu124–Ile130); B (Asn97–Gln107), B′ (Tyr117–Leu120), two which flank region Arg109–Met116, for no peaks are heteronuclear single quantum coherence spectrum. These would identify folding association initiation sites GED. Interestingly, they also coincide with helical state, suggesting that helix formation leads self-association