STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

The hake deepwater semi-pelagic ("pedra-bola") longline fishery in the Algarve (southern Portugal)

A 2-year study of the European hake (Merluccius merluccius) semi-pelagic ("pedra-bola) longline fishery was carried out in the Algarve (southern Portugal). This fishery takes place on the continental slope at 200-700 m depths. using monofilament longlines that are lifted off the bottom at regular intervals by glass balls. Hook selectivity trials were carried out with four hook sizes (SIAPAL brand numbers 10, 9, 7 and 5) from March to August 1997 and May to August 1998. At least 32 species of fish and invertebrates were caught, with hake dominating the catch (41 and 45% of the catch in numbers), followed by Galeus melastomus (23 and 19%), Micromesistius poutassou (10 and 7%), Benthodesmus elongatus (8 and 5%), Etmopterus pusillus (6 and 9%) and Scyliorhinus canicula (5 and 4%). Apart from the hake, and some species of commercial value such as G. melastomus (only the large individuals), Ray's bream (Brama brama, 1.4 and 4%), silver scabbard fish (Lepidopus caudatus. 1 and 2.5%), swordfish (Xiphins gladius, <1%), wreckfish (Polyprion americanus, <1%). conger eel (Conger conger. <1%), and bluemouth rockfish (Helicolenus dactylopterus, <1%)- most of the other species are discarded, used as bait in traps or consumed by the fishermen. Catch rates (number of fish per 100 hooks) for hake and for all species combined decreased significantly with hook size. Hake catch size frequency distributions for the different hook sizes in 1997 and 1998 were highly overlapped, with the four different hooks catching a wide range of sizes. Although catch size frequency distributions for the different sized hooks were not significantly different in 1997, hake caught in 1998 were characterised by smaller sizes and size ranges. (C) 2001 Elsevier Science B.V. All rights reserved.