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

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

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

J Mol Biol. 2011 Feb 4;405(5):1202-14

Authors: Chakraborty S, Hosur RV

GTPase effector domain (GED) of dynamin forms megadalton-sized assembly in vitro, rendering its structural characterization highly challenging. To probe the core of the GED assembly, we performed H/D exchange in native state and analyzed the residual amides following dissociation by dimethyl sulfoxide (DMSO). The data indicated a hierarchy in solvent exposure: Ser2-Glu13, Glu23-Phe32, Asp37-Gln43, Val51-Met55, and Lys60-Asp64 followed by the remaining segments. This reflects the chain packing in the core of the assembly. The segment Leu65-Pro138 in the C-terminal half is largely in the interior of the core, while the N-terminal segment Asp37-Asp64 traverses into and out of the core. Next, we characterized the structural and motional behavior of the DMSO-denatured state. The stretches Gly9-Lys18, Asp37-Arg42, Lys68-Met74, and Ser136-Thr137 were seen to display alternate conformations in slow exchange. In the major population, both ? and ? propensities were seen along the polypeptide chain. Spectral density analysis of (15)N R(1), R(2), and (1)H-(15)N nuclear Overhauser effect collected at 600 and 800*MHz suggested the presence of four domains of slow motions, namely, A (Leu40-Tyr91), A' (Leu124-Ile130); B (Asn97-Gln107), B' (Tyr117-Leu120), two of which flank the region Arg109-Met116, for which no peaks are seen in the heteronuclear single quantum coherence spectrum. These domains would identify folding and association initiation sites of GED. Interestingly, they also coincide with the helical domain in the native state, suggesting that helix formation leads to self-association of GED.

PMID: 21144852 [PubMed - indexed for MEDLINE]



Source: PubMed