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

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 15N-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.

Structural and Functional Consequences of the Weak Binding of Chlorin e6 to Bovine Rhodopsin.

The chlorophyll-derivative chlorin e6 (Ce6) identified in the retinas of deep-sea ocean fish is proposed to play a functional role in red bioluminescence detection. Fluorescence and 1 H NMR spectroscopy studies with the bovine dim-light photoreceptor, rhodopsin, indicate that Ce6 weakly binds to it with µm affinity. Absorbance spectra prove that red light sensitivity enhancement is not brought about by a shift in the absorbance maximum of rhodopsin. 19 F NMR experiments with samples where 19 F labels are either placed at the cytoplasmic binding site or incorporated as fluorinated retinal indicate that the cytoplasmic domain is highly perturbed by binding, while little to no changes are detected near the retinal. Binding of Ce6 also inhibits G-protein activation. Chemical shift changes in 1 H-15 N NMR spectroscopy of 15 N-Trp labeled bovine rhodopsin reveal that Ce6 binding perturbs the entire structure. These results provide experimental evidence that Ce6 is an allosteric modulator of rhodopsin.

Characterization of Denatured States and Reversible Unfolding of Sensory Rhodopsin II.

Our understanding on the folding of membrane proteins lags behind that of soluble proteins due to challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding pathways, we have systematically investigated the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 ΧSDS), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal-binding pocket is disrupted, with transmembrane residues becoming more solvent exposed. Folding of pSRII from an SDS-denatured state harboring a covalently bound retinal chromophore shows deviations from an apparent two-state behavior. SDS denaturation to form the sensory opsin apo-protein is reversible. We report pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins.

Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy.

Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal 'snapshots' of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination and protein labelling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analysing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.