Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence

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Abstract
The effects of guanine nucleotides on the intrinsic and extrinsic fluorescence properties of dynamin were assessed. The intrinsic Trp (tryptophan) fluorescence spectra of purified recombinant dynamin-1 and -2 were very similar, with a maximum at 332 nm. Collisional quenching by KI was weak (∼30%), suggesting that the majority of Trp residues are buried. Binding of guanine nucleotides decreased intrinsic fluorescence by 15–20%. Titration of the effects showed that GTP and GDP bound to a single class of non-interacting sites in dynamin tetramers with apparent dissociation constants (Kd) values of 5.4 and 7.4 μM (dynamin-1) and 13.2 and 7.1 μM (dynamin-2) respectively. Similar dissociation constant values for both nucleotides were obtained by titrating the quenching of IAEDANS [N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine]-labelled dynamin-2. Despite the similar binding affinities, GTP and GDP result in different conformations of the protein, as revealed by sensitivity to proteinase K fragmentation. Dynamins contain five Trp residues, of which four are in the PH domain (pleckstrin homology domain) and one is in the C-terminal PRD (proline/arginine-rich domain). Guanine nucleotides quenched fluorescence emission from a truncated (ΔPRD) mutant dynamin-1 to the same extent as in the full-length protein, suggesting conformational coupling between the G (groove)-domain and the PH domain. Efficient resonance energy transfer from PH domain Trp residues to bound mant-GTP [where mant stands for 2′-(3′)-O-(N-methylanthraniloyl)] suggests that the G-domain and PH domain are in close proximity (5–6 nm). Promotion of dynamin-2 oligomerization, by reduction in ionic strength or increasing protein concentration, had little effect on intrinsic dynamin fluorescence. However, fluorescence emission from IAEDANS·dynamin-2 showed a significant spectral shift on oligomerization. In addition, energy transfer was observed when oligomerization was promoted in mixtures of IAEDANS·dynamin-2 and 4-(4-dimethylaminophenylazo)benzoic acid-coupled dynamin-2, an effect that was counteracted by GTP but not GDP.