The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor. © 2009 Nature America, Inc. All rights reserved

Modeling allosteric signal propagation using protein structure networks

Allosteric communication in proteins can be induced by the binding of effective ligands, mutations or covalent modifications that regulate a site distant from the perturbed region. To understand allosteric regulation, it is important to identify the remote sites that are affected by the perturbation-induced signals and how these allosteric perturbations are transmitted within the protein structure. In this study, by constructing a protein structure network and modeling signal transmission with a Markov random walk, we developed a method to estimate the signal propagation and the resulting effects. In our model, the global perturbation effects from a particular signal initiation site were estimated by calculating the expected visiting time (EVT), which describes the signal-induced effects caused by signal transmission through all possible routes. We hypothesized that the residues with high EVT values play important roles in allosteric signaling. We applied our model to two protein structures as examples, and verified the validity of our model using various types of experimental data. We also found that the hot spots in protein binding interfaces have significantly high EVT values, which suggests that they play roles in mediating signal communication between protein domains

Defeating Alzheimer's disease and other dementias: a priority for European science and society

Alzheimer’s disease (AD) is the leading cause of dementia, and because the primary risk factor for AD is old age, the prevalence of the disease is increasing dramatically with ageing populations worldwide. Even in high-income countries, the cost of medical care and associated societal burdens of dementia threaten to become overwhelming as more people live into old age. In view of the lack of progress in developing a cure for AD and the rapidly increasing costs of dementia, policy makers and governments have a powerful incentive to provide more resources to develop AD therapeutics. The Lancet Neurology Commission was formed with the overarching aim to provide information and expert recommendations to policy makers and political leaders about the growing problem of AD and related dementias of ageing. The past two decades have seen remarkable improvements in the quality of care for patients with AD, with a research-driven shift to more personalised and integrated team-oriented care. Epidemiological and genetic studies have identifi ed many factors that increase the risk of AD. Prevention studies have highlighted the possibility of targeting risk and protective factors to delay onset, with the promise of reducing the overall prevalence of dementia. However, no treatment is yet available to halt or reverse the underlying pathology of established AD. Indeed, an eff ective therapy for AD is perhaps the greatest unmet need facing modern medicine. Basic biomedical research has provided insights into the causes and pathogenesis of AD and other neurodegenerative diseases, but improved understanding of disease mechanisms will be needed to develop safe and eff ective disease-modifying treatments. Nonetheless, several drugs are currently in late phases of clinical development. The Commission considered a range of challenges that need to be addressed to reduce the burden of dementia, and these challenges are discussed in detail in the main sections of our report: health economics (section 1), epidemiology (section 2), prevention (section 3), genetics (section 4), biology (section 5), diagnosis (section 6), treatment (sections 7, 8), care (section 9), and ethics (section 10). In panel 1 we summarise the key fi ndings of the Commission, with recommendations about how patient care and related research—from basic to clinical— in AD and other dementias should be organised in the future. A concerted eff ort to tackle dementia is needed, with a substantial overall increase in government and private investment in the care of patients and the search for AD therapeutics. Europe is well placed to take the world lead, in partnership with international organisations, to develop new approaches to prevent or cure AD and other dementias and to provide models of compassionate care for patients. As the cost of care increases, funds must not be shunted from basic research, clinical research, and drug-discovery programmes. In fact, a substantial increase in long-term funding for multidisciplinary research programmes is absolutely essential to reduce the burden of individual suff ering and the enormous societal cost of AD. Only targeted increases in research investment will provide any hope of fi nding a cure for AD or developing strategies to delay the onset or slow the progression of the disease

Opsin is a phospholipid flippase

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ < 10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop. © 2011 Elsevier Ltd All rights reserved.link_to_subscribed_fulltex