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The Dna J homologue, auxilin, acts as a co-chaperone for Hsc70 in the uncoating of clathrin-coated vesicles during endocytosis. Biochemical studies have aided understanding of the uncoating mechanism but until now there was no structural information on how auxilin interacts with the clathrin cage. Here we have determined the three-dimensional structure of a complex of auxilin with clathrin cages by cryo-electron microscopy and single particle analysis. We show that auxilin forms a discrete shell of density on the inside of the clathrin cage. Peptide competition assays confirm that a candidate clathrin box motif in auxilin, LLGLE, can bind to a clathrin construct containing the beta-propeller domain and also displace the well-characterised LLNLD clathrin box motif derived from the beta-adaptin hinge region. The means by which auxilin could both aid clathrin coat assembly and displace clathrin from AP2 during uncoating is discussed.

Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL-GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL-GroES. Surprisingly, trans-only also folded smaller GroEL-GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a trans-only complex rescued a GroEL-deficient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL-GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more efficient folding.

Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-beta structure. However, there is a lack of information relating the 4.8 A beta-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-beta ribbon is composed of relatively flat beta-sheets rather than being the highly twisted, beta-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in beta-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.

The chaperonin GroEL drives its protein-folding cycle by cooperatively binding ATP to one of its two rings, priming that ring to become folding-active upon GroES binding, while simultaneously discharging the previous folding chamber from the opposite ring. The GroEL-ATP structure, determined by cryo-EM and atomic structure fitting, shows that the intermediate domains rotate downward, switching their intersubunit salt bridge contacts from substrate binding to ATP binding domains. These observations, together with the effects of ATP binding to a GroEL-GroES-ADP complex, suggest structural models for the ATP-induced reduction in affinity for polypeptide and for cooperativity. The model for cooperativity, based on switching of intersubunit salt bridge interactions around the GroEL ring, may provide general insight into cooperativity in other ring complexes and molecular machines.

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane., (Copyright 2001 Academic Press.)

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis., (Copyright 2001 Academic Press.)

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein., (Copyright 2001 Academic Press.)

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map., (Copyright 2001 Academic Press.)

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

In this paper we describe reconstructions by electron cryo-microscopy of two oligomeric states of the pore-forming toxin pneumolysin. The results are interpreted by the fitting of atomic models of separated domains to the 3-dimensional electron density maps, revealing two steps in the mechanism of pore formation by the family of cholesterol-binding toxins. We briefly describe the observation of the toxin pore in model membranes and contrast the apparent mechanism of pneumolysin with that of other pore-forming toxins.

Recent advances in transmission electron microscopy (EM) hardware, low-temperature methods and image-processing software have made cryo-EM an important complement to X-ray crystallography and NMR for macromolecular structure determination, particularly of large assemblies. This review provides a summary of the main advances and a survey of the capabilities of this approach.

Biological processes involving movement, such as muscle contraction or the opening of an ion channel through a membrane, are mediated through conformational changes. These changes often occur in large and flexible macromolecular complexes. Cryo-electron microscopy provides a means of capturing different conformational states of such assemblies. Even if the resulting density maps are at low resolution, they can be combined with atomic structures of subcomplexes or isolated components determined by X-ray crystallography or NMR. This review presents a brief summary of the principles and recent advances in macromolecular structure determination by cryo-electron microscopy.

Three conformations of the thermosome, an archaeal group II chaperonin, have been determined by cryo-electron microscopy (EM). We describe an open form of the double-ring oligomer, a closed form and a bullet-shaped form with one ring open and the other closed. Domain movements have been deduced by docking atomic coordinates into the EM maps. The subunit apical domains, bearing the putative substrate binding sites, rotate about 30 degrees upwards and twist in the plane of the ring from the closed to the open conformation. The closed rings have their nucleotide binding pockets closed by the intermediate domains, but in the open rings, the pocket is accessible., (Copyright 2000 Academic Press.)

The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily., (Copyright 2000 Academic Press.)

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.

The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells., (Copyright 1999 Academic Press.)

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-beta-scaffold, with beta-strands perpendicular and beta-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3'-kinase, using cryo-electron microscopy and image processing at 25 A resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of approximately 600 A and an axial subunit repeat of approximately 27 A. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20x40-A protofilaments can only accommodate one pair of flat beta-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements. Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES. Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 A resolution for GroEL and GroEL-GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP. Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein-binding sites. We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

We present characterization of the rhodopsin, Gq and phosphatidylinositol-specific phospholipase C (PLC) from the signal transduction pathway of cephalopod photoreceptors. Cephalopod rhodopsins are unique in possessing a C-terminal extension of proline-rich repeats, and they have a strong tendency to form ordered arrays. Two-dimensional arrays of a full-length and C-terminally-truncated cephalopod rhodopsin have been obtained. The C termini appear to cluster the rhodopsins into small groups. An AlF4(-)-activated Gq alpha subunit has been isolated and shown to activate a partially purified PLC beta. This 130 kDa PLC, isolated by absorption on heparin agarose, showed a specific activity of 195 nmol of phosphatidylinositol 4,5-bisphosphate hydrolysed per milligram of protein per minute in the presence of 1.6 microM free calcium.

Rhodopsin is the G-protein-coupled membrane receptor that initiates the visual transduction cascade in retinal photoreceptors. In the present study rhodopsin from the dark-adapted retinas of squid (Loligo forbesi) was detergent-extracted, purified, and reconstituted into native squid photoreceptor lipids following proteolytic cleavage of its prolinerich C-terminus. Two-dimensional crystals of C-terminally truncated rhodopsin reconstituted from octyl glucoside solution formed in a p222(1) lattice (a = 44 A, b = 131 A). Electron micrographs of frozen-hydrated crystals were processed and a projection structure to 8 A resolution was calculated. The projection map obtained is very similar to maps previously determined for bovine and frog rhodopsins although the crystal packing of the molecules is quite different. Comparison of the maps shows that the arrangement of alpha-helices in the proteins is very similar despite their great phylogenetic distance; this structure is likely to be present in the whole superfamily of G-protein-coupled receptors. Invertebrate rhodopsins have a large insertion in the helix 5-helix 6 loop. Assignment of an additional density in the squid rhodopsin map to this region supports a previously proposed helix assignment and identifies the end-to-end contacts as helices 1 and 5.

Chaperonins form large, cage-like structures that act as protein folding machines. Recent developments in electron cryo-microscopy and image processing are helping to reveal the mechanism of chaperonin-mediated protein folding.

The chaperonin GroEL is a large, double-ring structure that, together with ATP and the cochaperonin GroES, assists protein folding in vivo. GroES forms an asymmetric complex with GroEL in which a single GroES ring binds one end of the GroEL cylinder. Cross-linking studies reveal that polypeptide binding occurs exclusively to the GroEL ring not occupied by GroES (trans). During the folding reaction, however, released GroES can rebind to the GroEL ring containing polypeptide (cis). The polypeptide is held tightly in a proteolytically protected environment in cis complexes, in the presence of ADP. Single turnover experiments with ornithine transcarbamylase reveal that polypeptide is productively released from the cis but not the trans complex. These observations suggest a two-step mechanism for GroEL-mediated folding. First, GroES displaces the polypeptide from its initial binding sites, sequestering it in the GroEL central cavity. Second, ATP hydrolysis induces release of GroES and productive release of polypeptide.

We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40-50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4-domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.

Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.

Background: The chaperonins, a family of molecular chaperones, are large oligomeric proteins that bind nonnative intermediates of protein folding. They couple the release and correct folding of their ligands to the binding and hydrolysis of ATP. Chaperonin 60 (cpn60) is a decatetramer (14-mer) of 60 kD subunits. Folding of some ligands also requires the cooperation of cpn10, a heptamer of 10 kD subunits., Results: We have determined the three-dimensional arrangements of subunits in Rhodobacter sphaeroides cpn60 in the nucleotide-free and ATP-bound forms. Negative stain electron microscopy and tilt reconstruction show the cylindrical structure of the decatetramer comprising two rings of seven subunits. The decatetramer consists of two cages joined base-to-base without a continuous central channel. These cages appear to contain bound polypeptide with an asymmetric distribution between the two rings. The two major domains of each subunit are connected on the exterior of the cylinder by a narrower bridge of density that could be a hinge region. Binding of ATP to cpn60 causes a major rearrangement of the protein density, which is reversed upon the hydrolysis of the ATP. Cpn10 binds to only one end of the cpn60 structure and is visible as an additional layer of density forming a cap on one end of the cpn60 cylinder., Conclusions: The observed rearrangement is consistent with an inward 5-10 degrees rotation of subunits, pivoting about the subunit contacts between the two heptamers, and thus bringing cpn60 domains towards the position occupied by the bound polypeptide. This change could explain the stimulation of ATPase activity by ligands, and the effects of ATP on lowering the affinity of cpn60 for ligands and on triggering the release of folding polypeptides.

Treatment of outer segment membranes from Loligo forbesi with endoprotease-V8 from Staphylococcus aureus results in cleavage of the C-terminal extension of the squid rhodopsin, with accompanying reduction of the apparent molecular weight from 47,000 to 36,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Negative-stain electron microscopy of the intact membranes shows that small clusters of the rhodopsin C-termini form structures extending from the membrane surface and that these are absent after protease treatment. Fourier transform infrared spectra of the amide I band of the protein indicate that removal of the C-terminal extension increases the relative alpha-helical content of squid rhodopsin to a level comparable to that for bovine rhodopsin in disk membranes, and to an extent which suggests that the alpha-helical structure lies mainly in the M(r) 36,000 (transmembrane) section of the protein. Saturation-transfer electron spin resonance (ESR) spectroscopy of the spin-labeled protein reveals that the rotational diffusion of squid rhodopsin in outer segment membranes that have been extensively washed with urea to remove peripheral proteins is much slower than that of bovine rhodopsin in rod outer segment disk membranes. This reduction in rotational mobility is also found with purified squid rhodopsin reconstituted in egg phosphatidylcholine and in urea-washed outer segment membranes which have been treated with endoprotease-V8 to remove the C-terminal extension of squid rhodopsin. In the latter case, the saturation-transfer ESR spectra are virtually identical to those of the non-proteolyzed membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The virus-like particles (VLPs) of the yeast retrotransposon Ty are genetically, structurally and functionally analogous to retroviral nucleocapsids or cores. Like retroviral cores Ty-VLPs package and possibly promote the enzyme activities for reverse transcription and integration, as well as encapsulating the RNA that is the intermediate in retrotransposition. Here we show that Ty-VLPs assemble into symmetrical structures across a broad distribution of particle sizes. This spread of sizes violates the principle of quasi-equivalent packing. In addition, RNase accessibility experiments suggest that these particles form an open structure that does not protect the encapsulated RNA. These features distinguish Ty-VLPs from typical spherical viral capsids in both structure and function.

The sequence of squid (Loligo forbesi) rhodopsin was determined by protein and cDNA sequencing. The protein has close similarity to octopus rhodopsin, having an N-terminal region (residues 1-340) which resembles other guanine-nucleotide-binding protein (G-protein)-linked receptors and a repetitive proline-rich C-terminus (residues 340-452). Comparison of the sequence of squid rhodopsin with those of other members of the G-protein-linked receptor superfamily reveals features which we predict to have both structural and functional importance.

1. At least two distinct G-proteins are activated by light in squid photoreceptor membranes, a 45,000 mol. wt cholera toxin substrate and a 40,000 mol. wt pertussis toxin substrate. 2. The light-stimulated GTPase activity is partially inhibited by pretreatment with either toxin and abolished by treatment with both toxins. 3. At 24 degrees C, a free calcium ion concentration of 1 microM inhibits GTPase activity of both toxin substrates and ADP ribosylation by pertussis toxin. 4. This calcium sensitivity of squid G-proteins may be important in interpretation of experimental results on the phosphoinositide or other signalling pathways in squid visual transduction.

Light stimulates the hydrolysis of exogenous, [3H]inositol-labeled phosphatidylinositol bisphosphate (PtdInsP2) added to squid photoreceptor membranes, releasing inositol trisphosphate (InsP3). At free calcium levels of 0.05 microM or greater, hydrolysis of the labeled lipid is stimulated up to 4-fold by GTP and light together, but not separately. This activity is the biochemical counterpart of observations on intact retina showing that a rhodopsin-activated GTP-binding protein is involved in visual transduction in invertebrates, and that InsP3 release is correlated with visual excitation and adaptation. Using an in vitro assay, we investigated the calcium and GTP dependence of the phospholipase activity. At calcium concentrations between 0.1 and 0.5 microM, some hydrolysis occurs independently of GTP and light, with a light- and GTP-activated component superimposed. At 1 microM calcium there is no background activity, and hydrolysis absolutely requires both GTP and light. Ion exchange chromatography on Dowex 1 (formate form) of the water-soluble products released at 1 microM calcium reveals that the product is almost entirely InsP3. Invertebrate rhodopsin is homologous in sequence and function to vertebrate visual pigment, which modulates the concentration of cyclic GMP through the mediation of the GTP-binding protein transducin. While there is some evidence that light also modulates PtdInsP2 content in vertebrate photoreceptors, the case for its involvement in phototransduction is stronger for the invertebrate systems. The results reported here support the scheme of rhodopsin----GTP-binding protein----phospholipase C activation in invertebrate photoreceptors.

Frog retinal rod outer segments, oriented by a magentic field, were shown to contain rhodopsin alpha-helical segments preferentially aligned perpendicular to the plane of the disc membrane, by the technique of infrared linear dichroism. Infrared absorption parallel and perpendicular to the rod axes by peptide C parallel to O groups, whose absorption band contains alpha-helical and random coil components at slightly different frequencies, showed positive dichroism centered on the alpha-helix frequence. We conclude that the alpha-helical portion of the protein has an average orientation in the transmembrane direction. Furthermore, infrared spectra of rods in 2H2O Ringer's solution exhibit two distinct peptide amino group absorption bands: the unexchanged N-2H band, which is nondichroic. This implies that the oriented part of the protein is in the lipid bilayer, supporting a model for rhodopsin with a hydrophobic core containing partially oriented alpha-helices and hydrophilic ends consisting of unoriented polypeptide.

The activation of photoreceptor GTP-binding protein by rhodopsin was studied in squid photoreceptors and in crossreactions between the squid and bovine proteins. Turbidity changes were observed in the far-red after photoexcitation of rhodopsin with brief flashes and were used to probe interactions between photoreceptor membrane suspensions and soluble protein extracts. Our findings are squid photoreceptors contain a GTP-binding protein detectable by light- and GTP-sensitive turbidity changes and by limited sequence homology of a 46-kilodalton polypeptide to the alpha-subunit of bovine GTP-binding protein; the squid membranes activate bovine GTP-binding protein qualitatively in the same way as bovine rhodopsin; the 46-kilodalton component is present in a membrane-bound fraction but is more abundant in a crude, soluble fraction of squid rhabdomes, and this soluble fraction can interact with either squid or bovine rhodopsin-containing membranes; light-activated GTPase activities in all of these preparations are consistent with the light-induced turbidity changes. These results show that rhodopsin activation of GTP-binding protein is highly conserved in vertebrate and cephalopod photoreceptors. Since squid rhodopsin is immobilized in precisely ordered microvilli, this suggests that activation of GTP-binding protein in cephalopod photoreceptors occurs in the absence of rhodopsin diffusion. The rhodopsin immobility may be compensated by higher mobility of the soluble GTP-binding protein.

Photoreceptor outer segments isolated from squid retina are known to contain a light-activated GTP-binding protein. Here it is shown that these photoreceptors contain around 0.01 mol cyclic GMP per mol rhodopsin. Adding GTP in the dark stimulates the production of 0.0003-0.001 mol cyclic GMP/mol rhodopsin per min. GTP and light cause a 2-fold faster increase in cyclic GMP. These results show that either (1) squid rhodopsin activates a guanylate cyclase, or (2) there is a constant guanylate cyclase activity and photoexcited rhodopsin inhibits a cyclic GMP phosphodiesterase.