Biomedical subjects

O Llorca

Publications and source records attributed to O Llorca.

25 records · Page 2Linked to original sources

PubMed1997

Effects of the inter-ring communication in GroEL structural and functional asymmetry.

The chaperonin GroEL consists of a double-ring structure that assists protein folding in the presence of GroES and ATP. Recent studies suggest that the 7-mer ring is the functional unit where protein folding takes place. Nevertheless, both GroEL rings are required to complete the reaction cycle through signals transmitted between the two rings. Electron microscopy, image processing, and biochemical analysis of GroEL, a single-ring mutant (SR1) and a inter-ring communication affected mutant (A126V), in the presence of ATP and adenylyl imidodiphosphate, have allowed the identification of a conformational change in the apical domains that is strictly dependent on the communication between the two GroEL rings. It is deduced from these results that the binding of nucleotide to both GroEL rings generates, as a consequence of the inter-ring communication, a functionally and structurally asymmetric particle. This asymmetric particle has a ring with a small conformational change in its apical domains and high affinity toward unfolded substrate and GroES, and the other ring has a larger conformational change in its apical domains and lower affinity toward substrate and GroES.

Adenosine Triphosphate↗

PubMed1997

Symmetric GroEL-GroES complexes can contain substrate simultaneously in both GroEL rings.

Incubation of rhodanese with hche aperonins GroEL and GroES (1:2 GroEL14:GroES7 molar ratio) under functional and steady state conditions for ATP leads to the formation of a high proportion of rhodanese-bound symmetric complexes (GroEL14(GroES7)2), as revealed by native electrophoresis. Aliquots of such samples were observed under the electron microscope, and the symmetric particles were classified using neuronal networks and multivariate statistical analysis. Three different populations of symmetric particles were obtained which contained substrate in none, one or both GroEL cavities, respectively. The presence of substrate in the symmetric complexes under functional conditions supports their role as active intermediates in the protein folding cycle. These results also suggest that symmetric GroEL-GroES complexes can use both rings simultaneously for folding, probably increasing the efficiency of the reaction.

Adenosine Triphosphate↗

PubMed1997

Role of the amino terminal domain in GroES oligomerization.

Digestions of the GroES oligomer with trypsin, chymotrypsin and Glu-C protease from Staphylococcus aureus V8 (V8) have helped to locate three regions in the GroES sequence that are sensitive to limited proteolysis and have provided information of the GroES domains involved in monomer-monomer and GroEL interaction. The removal of the first 20 or 27 amino acids of the N-terminal region of each GroES monomer by trypsin or chymotrypsin respectively, abolish the oligomerization of the GroES complex and its binding to GroEL. The V8-treatment of GroES promotes the breakage of the peptide bond between Glu18 and Thr19 but not the liberation of the N-terminal fragment from the GroES oligomer, which is capable of forming with GroEL a complex active in protein folding. It is deduced from these results that the N-terminal region of the GroES monomer is involved in monomer-monomer interaction, providing experimental evidence that relates some biochemical properties of GroES with its three-dimensional structure at atomic resolution.

Amino Acid Sequence↗

PubMed1997

Conformational changes in the GroEL oligomer during the functional cycle.

The conformational changes that the GroEL oligomer undergoes upon nucleotide and cochaperonin GroES binding have been studied using electron microscopy and image processing techniques. Average side views of the three allosteric states (TT, TR, and RR, which correspond to none, one, or both of the two heptameric rings of the GroEL oligomer occupied by nucleotide, respectively) of GroEL and GroEL-GroES complexes for ADP, ATP, and two nonhydrolyzable analogs (AMP-PNP and ATP gamma S) have been obtained at 20-25 A resolution. Both AMP-PNP and ATP induce similar conformational shifts in the apical domains of GroEL. At the TR state, only one of the GroEL rings shows an upward and outward movement of the apical domains ("open state"). At the RR state for AMP-PNP and ATP, both GroEL rings undergo conformational changes, albeit of different magnitude, giving rise to a structurally asymmetric particle (one ring in the "open" state, while the other is in an "intermediate" state). These changes are also observed when GroEL is incubated with ADP and Pi, but not with ADP, which suggests that upon ATP binding, GroEL undergoes a conformational change that is partly maintained after ATP hydrolysis and as long as ADP and Pi are bound to the GroEL ring. The conformational changes undergone by GroEL are discussed within the framework of a proposed GroEL cycle mechanism.

Adenine Nucleotides↗

PubMed1996

Biochemical characterization of symmetric GroEL-GroES complexes. Evidence for a role in protein folding.

When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K+ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL:GroES oligomer ratio). The percentage of symmetric complexes present is directly related to the [ATP]/[ADP] ratio and to the K+ concentration. Kinetic analysis shows that there is a cycle of formation and disappearance of symmetric complexes. A correlation between the presence of symmetric complexes in the incubation mixture and its rhodanese folding activity suggests some active role of these complexes in the protein folding process. Accordingly, under functional conditions, symmetric complexes are found to contain denatured rhodanese. These data suggest that binding of substrate inside the GroEL cavity takes place before the symmetric complex is formed.

Chaperonin 10↗

PubMed1995

Prediction of the structure of GroES and its interaction with GroEL.

The three-dimensional structure of the GroES monomer and its interaction with GroEL has been predicted using a combination of prediction tools and experimental data obtained by biophysical [electron microscope (EM), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR)] and biochemical techniques. The GroES monomer, according to the prediction, is composed of eight beta-strands forming a beta-barrel with loose ends. In the model, beta-strands 5-8 run along the outer surface of GroES, forming an antiparallel beta-sheet with beta 4 loosely bound to one of the edges. beta-strands 1-3 would then be parallel and placed in the interior of the molecule. Loops 1-3 would face the internal cavity of the GroEL-GroES complex, and together with conserved residues in loops 5 and 7, would form the active surface interacting with GroEL.

Amino Acid Sequence↗

PubMed1994

The formation of symmetrical GroEL-GroES complexes in the presence of ATP.

The incubation of chaperonins cpn60 (GroEL) and cpn10 (GroES) from E. coli in the presence of Mg-ATP and KCl generates the formation, as revealed by electron microscopy, of GroEL-GroES complexes with a symmetrical shape in which one toroidal GroES oligomer is bound to each end of the tetradecameric GroEL aggregate (1:2 GroEL:GroES oligomer molar ratio). The symmetrical complexes are not observed in the presence of ADP or the non-hydrolyzable ATP analog, ATP gamma S, where only asymmetrical complexes (1:1 GroEL:GroES oligomer molar ratio) are formed. These results suggest that ATP hydrolysis is required for the formation of symmetrical complexes.

Adenosine Triphosphate↗