Chaperonin function: folding by forced unfolding.

The ability of the GroEL chaperonin to unfold a protein trapped in a misfolded condition was detected and studied by hydrogen exchange. The GroEL-induced unfolding of its substrate protein is only partial, requires the complete chaperonin system, and is accomplished within the 13 seconds required for a single system turnover. The binding of nucleoside triphosphate provides the energy for a single unfolding event; multiple turnovers require adenosine triphosphate hydrolysis. The substrate protein is released on each turnover even if it has not yet refolded to the native state. These results suggest that GroEL helps partly folded but blocked proteins to fold by causing them first to partially unfold. The structure of GroEL seems well suited to generate the nonspecific mechanical stretching force required for forceful protein unfolding.

Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding.

The Escherichia coli chaperonins GroEL and GroES facilitate protein folding in an adenosine triphosphate (ATP)-dependent manner. After a single cycle of ATP hydrolysis by the adenosine triphosphatase (ATPase) activity of GroEL, the bi-toroidal GroEL formed a stable asymmetric ternary complex with GroES and nucleotide (bulletlike structures). With each subsequent turnover, ATP was hydrolyzed by one ring of GroEL in a quantized manner, completely releasing the adenosine diphosphate and GroES that were tightly bound to the other ring as a result of the previous turnover. The catalytic cycle involved formation of a symmetric complex (football-like structures) as an intermediate that accumulated before the rate-determining hydrolytic step. After one to two cycles, most of the substrate protein dissociated still in a nonnative state, which is consistent with intermolecular transfer of the substrate protein between toroids of high and low affinity. A unifying model for chaperonin-facilitated protein folding based on successive rounds of binding and release, and partitioning between committed and kinetically trapped intermediates, is proposed.

Symmetric complexes of GroE chaperonins as part of the functional cycle.

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

GroEL structure: a new chapter on assisted folding.

The X-ray structure of GroEL puts future studies on a firm footing, but there's still much work to be done before chaperonin-assisted protein folding is understood.

Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 A resolution.

BACKGROUND: In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme. RESULTS: We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 A resolution. The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase. CONCLUSIONS: The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum.

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.

Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria.

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.

Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins.

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.

GroES and the chaperonin-assisted protein folding cycle: GroES has no affinity for nucleotides.

The E. coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins. GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold. On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested [Martin, et al., Nature, 366 (1993) 279-282]. We confirm the photolabeling of GroES with 8-azido-ATP. However, other proteins not known to contain nucleotide binding sites also became photolabeled suggesting that labeling is non-specific. Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected. We conclude that GroES has no nucleotide binding site.

On the distribution of ligands within the asymmetric chaperonin complex, GroEL14.ADP7.GroES7.

In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7.MgADP7.GroES7]-[GroEL7]) or trans (i.e. [GroEL7.MgADP7]-[GroEL7.GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228-233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.

An analytical electron microscopic investigation of precipitation in an Al-Cu-Zn-Mg-Ag alloy.

The distribution, morphology, chemistry, and crystallography of the precipitates formed during aging of an Al-Cu-Zn-Mg-Ag alloy have been studied using analytical transmission electron microscopy. The first precipitates to appear during aging at 150 degrees C were thin hexagonal-shaped plate-like precipitates which formed on the (111)Al planes. These precipitates had a face-centred orthorhombic crystal structure and their composition was essentially CuAl2 although they contained a trace of silver. At peak hardness the microstructure consisted of the plate-like precipitates on (111)Al planes and theta' precipitates on (100)Al planes. Overaging resulted in the precipitation of equilibrium theta, CuAl2, which exhibited a lath morphology and an orientation-relationship with the matrix (210)Al magnitude of (110)gamma; (001)Al misoriented from (001)gamma by approximately 6 degrees. Prolonged overaging at 250 degrees C resulted in the formation of cuboid-shaped Al5(Cu,Zn)6Mg2 precipitates which had a cubic crystal structure and a cube:cube orientation-relationship with the matrix.

Perceptual bias in pain: a switch looks closer when it will relieve pain than when it won't.

Pain is fundamental to survival, as are our perceptions of the environment. It is often assumed that we see our world as a read-out of the sensory information that we receive; yet despite the same physical makeup of our surroundings, individuals perceive differently. What if we "see" our world differently when we experience pain? Until now, the causal effect of experimental pain on the perception of an external stimulus has not been investigated. Eighteen (11 female) healthy volunteers participated in this randomised repeated-measures experiment, in which participants estimated the distance to a switch placed on the table in front of them. We varied whether or not the switch would instantly stop a stimulus, set to the participant's pain threshold, being delivered to their hand, and whether or not they were required to reach for the switch. The critical result was a strong interaction between reaching and pain [F(1,181)=4.8, P=0.03], such that when participants experienced pain and were required to reach for a switch that would turn off the experimental stimulus, they judged the distance to that switch to be closer, as compared to the other 3 conditions (mean of the true distance 92.6%, 95% confidence interval 89.7%-95.6%). The judged distance was smaller than estimates in the other 3 conditions (mean±SD difference >5.7%±2.1%, t(181) >3.5, P<0.01 for all 3 comparisons). We conclude that the perception of distance to an object is modulated by the behavioural relevance of the object to ongoing pain.

Limb-specific autonomic dysfunction in complex regional pain syndrome modulated by wearing prism glasses.

In unilateral upper-limb complex regional pain syndrome (CRPS), the temperature of the hands is modulated by where the arms are located relative to the body midline. We hypothesized that this effect depends on the perceived location of the hands, not on their actual location, nor on their anatomical alignment. In 2 separate cross-sectional randomized experiments, 10 (6 female) unilateral CRPS patients wore prism glasses that laterally shifted the visual field by 20°. Skin temperature was measured before and after 9-minute periods in which the position of one hand was changed. Placing the affected hand on the healthy side of the body midline increased its temperature (Δ°C=+0.47 ± 0.14°C), but not if prism glasses made the hand appear to be on the body midline (Δ°C=+0.07 ± 0.06°C). Similarly, when prism glasses made the affected hand appear to be on the healthy side of the body midline, even though it was not, the affected hand warmed up (Δ°C=+0.28 ± 0.14°C). When prism glasses made the healthy hand appear to be on the affected side of the body midline, even though it was not, the healthy hand cooled down (Δ°C=-0.30 ± 0.15°C). Friedman's analysis of variance and Wilcoxon pairs tests upheld the results (P<0.01 for all). We conclude that, in CRPS, cortical mechanisms responsible for encoding the perceived location of the limbs in space modulate the temperature of the hands.