G protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human beta2-adrenoceptor (beta2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the beta2-AR.

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a builtin lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.Chaperonins are ubiquitous and essential mediators of cellular protein folding that consist of 14-18 subunits arranged in two stacked rings [1][2][3][4] . Each ring encompasses a central chamber that accommodates non-native polypeptides. All chaperonins share a similar subunit architecture, consisting of three distinct domains: an ATP-binding equatorial domain, a distal apical domain harboring the polypeptide-binding sites and an intermediate hinge domain. A key feature of chaperonins is their ability to close their chamber and encapsulate the bound substrate, thus providing a protected environment for protein folding. Opening and closing of the folding chamber is controlled by a conformational cycle driven by ATP binding and hydrolysis. Despite overall structural similarities, there are substantial differences between the eubacterial chaperonins, such as GroEL from E. coli [2][3][4][5] , and the

The ␤ 2 adrenoreceptor (␤ 2 AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCRmediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified ␤ 2 AR. Here we show that upon catecholamine binding, ␤ 2 ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating G s but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating G s and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions. G protein-coupled receptors (GPCRs)1 represent the largest family of membrane proteins in the human genome. They are responsible for the majority of cellular responses to hormones and neurotransmitters and are the largest group of targets for drug discovery. GPCRs are remarkably versatile biological sensors, responding to a broad spectrum of chemical entities ranging in size from a single photon of light, to ions, to small organic compounds, to peptides and protein hormones (1).Rhodopsin has long been used as a model system for studying the structure and mechanism of activation of GPCRs. It is the only GPCR for which a high resolution structure is available (2). Light-induced conformational changes have been elucidated by a series of elegant biophysical studies (3-8). Electron paramagnetic resonance spectroscopy studies provide evidence that photoactivation of rhodopsin involves a rotation and tilting of transmembrane domain 6 (TM6) relative to TM3 (3). Light-induced conformational changes have also been observed in the cytoplasmic domain spanning TM1 and TM2 (6) and the cytoplasmic end of TM7 and helix 8 (5).Structural and biophysical studies on other GPCRs are more limited. Fluorescence spectroscopic analysis of ␤ 2 ARs labeled with environmentally sensitive fluorescent probes detects movement of both TM3 and TM6 upon agonist binding (9). More detailed analysis of conformational changes in TM6 of the ␤ 2 AR provide evidence for a rigid body motion similar to that observed upon activation of rhodopsin (10, 11). Additional support for movement of TM3 and TM6 in the ␤ 2 AR comes from zinc cross-linking studies (12) and chemical reactivity measurem...

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