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Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin Alpha-Subunit Upon Binding a Soluble Mimic of Light-Activated Rhodopsin

Published

Author(s)

D M. Brabazon, N G. Abdulaev, John Marino, K D. Ridge

Abstract

Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (Gt) is still unknown. Previous efforts on the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer Gt activating potential [Abdulaev et al. (2000) J. Biol. Chem. 275, 39354-39363]. To assess whether these designed mimics could provide a structural insight into the interaction between light-activated rhodopsin and Gt, the ability of an HPTRX fusion protein comprised of the second (CD) and third (EF) cytoplasmic loops (HPTRX/CDEF) to bind Gt α-subunit (Gta) peptides was examined using nuclear magnetic resonance (NMR) spectroscopy. Transfer NOESY (TrNOESY) experiments how that an 11 amino acid peptide corresponding to the carboxyl-terminus of Gta (GtP), as well as a high-affinity peptide analog, HAP1, binds to HPTRX/CDEF in the fast-exchange regime and undergoes similar, subtle structural changes at the extreme carboxyl-terminus. Observed TrNOEs suggest that both peptides when bound to HPTRX/CDEF adopt a reverse turn that is consistent with the C-cap structure that has been previously reported for the interaction of GtP with the light-activated signaling state, metarhodopsin II (MII). In contrast, TrNOESY spectra provide no evidence for structuring of the amino terminus of either GtP or HAP1when bound to HPTRX/CDEF, nor do the spectra show any measurable changes in the CD and EF loop resonances of HPTRX/CDEF, which are conformationally dynamic and significantly exchange broadened. Taken together, the NMR observations indicate that HPTRX/CDEF, previously identified as a functional mimic of MII, is also an approximate structural mimic for this light-activated state of rhodopsin
Citation
Biochemistry
Volume
42
Issue
2

Keywords

G-protein, membrane protein, NMR, rhodopsin, signal transduction, thioredoxin, vision

Citation

Brabazon, D. , Abdulaev, N. , Marino, J. and Ridge, K. (2003), Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin Alpha-Subunit Upon Binding a Soluble Mimic of Light-Activated Rhodopsin, Biochemistry (Accessed April 18, 2024)
Created January 20, 2003, Updated October 12, 2021