Tai-Yang Kim

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the “phosphate-sensor” of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Characterization of membrane protein non-native states. 2. The SDS-unfolded states of rhodopsin

Little is known about the molecular nature of residual structure in unfolded states of membrane proteins. A screen of chemical denaturants to maximally unfold the mammalian membrane protein and prototypic G protein coupled receptor rhodopsin, without interference from aggregation, described in an accompanying paper (DOI 10.1021/bi100338e ), identified sodium dodecyl sulfate (SDS), alone or in combination with other chemicals, as the most suitable denaturant. Here, we initiate the biophysical characterization of SDS-denatured states of rhodopsin. Using absorption, steady-state and time-resolved fluorescence spectroscopy, dynamic light scattering, and cysteine accessibility studies, tertiary structure of denatured states was characterized. In agreement with the pattern of secondary structure changes detected by circular dichroism described in the accompanying paper (DOI 10.1021/bi100338e ), tertiary structure changes are distinct over four SDS concentration ranges based on the expected predominant micellar structures. Dodecyl maltoside (DM)/SDS mixed micelle spheres (0.05-0.3% SDS) turn into SDS spheres (0.3-3% SDS) that gradually (3-15% SDS) become cylindrical (above 15% SDS). Denatured states in SDS spheres and cylinders show a relatively greater burial of cysteine and tryptophan residues and are more compact as compared to the states observed in mixed micellar structures. Protein structural changes at the membrane/water interface region are most prominent at very low SDS concentrations but reach transient stability in the compact conformations in SDS spheres. This is the first experimental evidence for the formation of a compact unfolding intermediate state with flexible surface elements in a membrane protein.

Picosecond Multidimensional Fluorescence Spectroscopy: A Tool to Measure Real-time Protein Dynamics During Function †

Advanced multidimensional time‐correlated single photon counting (mdTCSPC) and picosecond time‐resolved fluorescence in combination with site‐directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico‐ to nanoseconds time scale. Time‐resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light‐activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC‐module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.

Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues.

The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein-ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled μ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.

Monitoring the Interaction of a Single G-Protein Key Binding Site with Rhodopsin Disk Membranes upon Light Activation

Heterotrimeric G-proteins interact with their G-protein-coupled receptors (GPCRs) via key binding elements comprising the receptor-specific C-terminal segment of the alpha-subunit and the lipid anchors at the alpha-subunit N-terminus and the gamma-subunit C-terminus. Direct information about diffusion and interaction of GPCRs and their G-proteins is mandatory for an understanding of the signal transduction mechanism. By using single-particle tracking, we show that the encounters of the alpha-subunit C-terminus with the GPCR rhodopsin change after receptor activation. Slow as well as less restricted diffusion compared to the inactive state within domains 60-280 nm in length was found for the receptor-bound C-terminus, indicating short-range order in rhodopsin packing.

Activation and molecular recognition of the GPCR rhodopsin – Insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.

Dissection of Environmental Changes at the Cytoplasmic Surface of Light‐activated Bacteriorhodopsin and Visual Rhodopsin: Sequence of Spectrally Silent Steps†

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100, 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M‐intermediate as well as the active metarhodopsin‐II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin‐II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin‐II decay.

Application of Single Molecule Fluorescence Microscopy to Characterize the Penetration of a Large Amphiphilic Molecule in the Stratum Corneum of Human Skin

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

A simulation-guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase.

Fluorescence correlation spectroscopy (FCS) is a single molecule based technique to temporally resolve rate-dependent processes by correlating the fluorescence fluctuations of individual molecules traversing through a confocal volume. In addition, chemical processes like protonation or intersystem crossing can be monitored in the sub-microsecond range. FCS thereby provides an excellent tool for investigations of protonation dynamics in proton pumps like cytochrome c oxidase (CcO). To achieve this, the pH-dependent fluorescent dye fluorescein was attached as a protonation probe to the CcO surface via site-specific labeling of single reactive cysteines that are located close to the entry point of a proton input channel (K-pathway). The analysis of protonation dynamics is complicated by overlapping triplet and protonation rates of the fluorophore. A Monte Carlo simulation based algorithm was developed to facilitate discrimination of these temporally overlapping processes thus allowing for improved protonation reaction rate determination. Using this simulation-guided approach we determined precise local proton association and dissociation rates and provide information about protein surface effects, such as proton collecting antennae, on the transport properties of proton transfer channels.