Fast Ion-Beam Inactivation of Viruses, Where Radiation Track Structure Meets RNA Structural Biology.

Here we show an interplay between the structures present in ionization tracks and nucleocapsid RNA structural biology, using fast ion-beam inactivation of the severe acute respiratory syndrome coronavirus (SARS-CoV) virion as an example. This interplay could be a key factor in predicting dose-inactivation curves for high-energy ion-beam inactivation of virions. We also investigate the adaptation of well-established cross-section data derived from radiation interactions with water to the interactions involving the components of a virion, going beyond the density-scaling approximation developed previously. We conclude that solving one of the grand challenges of structural biology - the determination of RNA tertiary/quaternary structure - is linked to predicting ion-beam inactivation of viruses and that the two problems can be mutually informative. Indeed, our simulations show that fast ion beams have a key role to play in elucidating RNA tertiary/quaternary structure.

A novel cause of rebreathing carbon dioxide related to removed CLIC-seal on the Dräger Apollo© anesthesia machine.

We present a case report involving two sequential, surgically uneventful, laparoscopic cholecystectomies using the same anesthesia machine (Drager Apollo©) for which the level of inspired carbon dioxide was noted to be elevated following various diagnostic interventions including replacing the sodalime, increasing fresh gas flows, and a full inspection of equipment for malfunction. Eventually it was discovered that a rubber ring seal connecting the Dragersorb CLIC system© to the sodalime canister was inadvertently removed during the initial canister exchange resulting in an apparent bypassing of the absorbent and thus an inability of the exhaled gas to contact the sodalime. To our knowledge this is the first such description of this potential cause of elevated inspired carbon dioxide and should warrant consideration when other conventional interventions have failed.

Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy.

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.

ATP-bound states of GroEL captured by cryo-electron microscopy.

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.

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

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.

The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL.

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.

Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy.

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.

ATP induces large quaternary rearrangements in a cage-like chaperonin structure.

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.