title: Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence.
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 (approximately 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 (K(d)) values of 5.4 and 7.4 microM (dynamin-1) and 13.2 and 7.1 microM (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 (DeltaPRD) 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.
authors.affiliation: Department of Neurobiology, Pharmacology and Physiology, University of Chicago, 947 E. 58th St., Chicago, IL 60637, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Solomaha
authors.forename: H Clive
authors.initials: HC
authors.lastname: Palfrey
mesh_terms: D000818:Animals; D034284:Dynamin I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D034285:Dynamin II / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D034284:Dynamin I; D034285:Dynamin II