Characterisation of denatured states of sensory rhodopsin II by solution-state NMR

Abstract

Sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis, is a novel model system for membrane protein folding studies. Recently, the SDS-denatured states and the kinetics for reversible unfolding of pSRII have been investigated, opening the door to the first detailed characterisation of denatured states of a membrane protein by solution-state nuclear magnetic resonance (NMR) using uniformly 15 N-labelled pSRII. SDS denaturation and acid denaturation of pSRII both lead to fraying of helix ends but otherwise small structural changes in the transmembrane domain, consistent with little changes in secondary structure and disruption of the retinal-binding pocket and tertiary structure. Widespread changes in the backbone amide dynamics are detected in the form of line broadening, indicative of µs-to-ms timescale conformational exchange in the transmembrane region. Detailed analysis of chemical shift and intensity changes lead to high-resolution molecular insights on structural and dynamics changes in SDS-and acid-denatured pSRII, thus highlighting differences in the unfolding pathways under the two different denaturing conditions. These results will form the foundation for furthering our understanding on the folding and unfolding pathways of retinal-binding proteins and membrane proteins in general, and also for investigating the importance of ligand-binding in the folding pathways of other ligand-binding membrane proteins, such as GPCRs. Highlights • We report detailed NMR studies on a denatured polytopic α-helical membrane protein • SDS-and acid-denatured pSRII experience small structural changes in the TM domains • Denatured pSRII undergoes widespread µs-to-ms timescale conformational exchange • High-resolution structural differences between SDS vs. acid denaturation are shown • Retinal influences the exchange equilibria amongst different conformations of pSRII Keywords Membrane proteins; Folding; Transmembrane helices; Chemical shifts; Backbone amides Abbreviations 7TM Seven transmembrane Χ SDS Mole fraction of SDS BEST Band-selective excitation short transient c7-DHPC Diheptanoylphosphatidylcholine; 1,2-diheptanoyl-sn-glycero-3-phosphocholine CD Circular dichroism H-bond Hydrogen bond HSQC Heteronuclear single quantum correlation MRE Mean residue ellipticity NMR Nuclear magnetic resonance pKa Negative base-10 logarithm of the acid dissociation constant pSRII Natronomonas pharaonis sensory rhodopsin II SDS Sodium dodecyl sulphate TM Transmembrane TROSY Transverse relaxation optimised spectroscopy TSP Trimethylsilylpropanoic acid UV/vis UV/visible

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

“…Moreover, order parameters of Ile and Leu tend to decrease with increasing distance to the retinal binding site (Figure S13), supporting the view that bound retinal confers conformational stability to the protein center, which is propagated by allosteric pathways to the outside of the bundle. This observation agrees with recent folding studies of sensory rhodopsin using solution NMR methods that indicated that the retinal binding region constitutes the folding core of the protein [68] . Similarly, Reat et al [69] .…”

“…The fact that we assigned the vast majority of observed peaks indicates that the missing resonances are broadened beyond detection by conformational exchange. Since the two isomeric forms that are present in the dark‐adapted state of bR interconvert too slowly to produce the observed line‐broadening, [68] we suspect that the line‐broadening is rather due to motional adaptations in the extracellular cavity of bR. We speculate that those processes might be related to the exchange of internal and bulk water [70] or the lack of stabilizing contacts in the bR monomer.…”

“…Moreover,o rder parameters of Ile and Leu tend to decrease with increasing distance to the retinal binding site ( Figure S13), supporting the view that bound retinal confers conformational stability to the protein center, which is propagated by allosteric pathways to the outside of the bundle.This observation agrees with recent folding studies of sensory rhodopsin using solution NMR methods that indicated that the retinal binding region constitutes the folding core of the protein. [68] Similarly,R eat et al [69] could demonstrate higher rigidity for moieties in the retinal-binding region using elastic incoherent neutron scattering.S urprisingly,n oI LV-methyl groups are present in the inside of the helix bundle at the extracellular side of bR, where 7crystallographic water molecules are found. Instead, aromatic and polar/charged residues mainly populate this cavity.…”