PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 12507429

Directed evolution of substrate-optimized GroEL/S chaperonins.

Jue D Wang·Christophe Herman·Kimberly A Tipton·Carol A Gross·Jonathan S Weissman
View original source ↗Export metadata ↓

Abstract

GroEL/S chaperonin ring complexes fold many unrelated proteins. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP). Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle. These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.

Explore related subjects

Adenosine TriphosphatasesChaperonin 10Chaperonin 60Directed Molecular EvolutionEscherichia coliEukaryotic CellsEvolution, MolecularGene Expression Regulation, BacterialGreen Fluorescent ProteinsLuminescent ProteinsMutationProkaryotic CellsProtein FoldingProtein IsoformsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Jue D Wang, Christophe Herman, Kimberly A Tipton, Carol A Gross, Jonathan S Weissman. 2002-12-27. Directed evolution of substrate-optimized GroEL/S chaperonins.. https://doi.org/10.1016/s0092-8674(02)01198-4

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2008

Isolation and characterization of Plasmodium falciparum UAP56 homolog: evidence for the coupling of RNA binding and splicing activity by site-directed mutations.

UAP56 (U2AF65 associated protein) is a member of the DEAD-box helicase family. Helicases are essential enzymes generally involved in the metabolism of nucleic acids. The gene encoding a member of DEAD-box family was cloned and characterized from the human malaria parasite Plasmodium falciparum. PfU52 is homologous to UAP56 and contains the RNA-dependent ATPase, RNA helicase and RNA binding activities. Using the parasite extract we report that PfU52 is involved in splicing reaction. Site-directed mutagenesis studies indicate that the conserved residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this activity is required for the enzymatic activities of PfU52. PfU52 is expressed in all the intraerythrocytic developmental stages of the parasite. In the present study we have reported the detailed characterization of PfU52 from P. falciparum and these results advance the knowledge regarding the function of UAP56 in general.

Adenosine Triphosphatases↗
PubMed2007

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells.

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.

Adenosine Triphosphatases↗
PubMed2007

Green tea catechins inhibit bacterial DNA gyrase by interaction with its ATP binding site.

Catechins are the main ingredients of green tea extracts and have been shown to possess versatile biological activities, including antimicrobial. We determined that the catechins inhibit bacterial DNA gyrase by binding to the ATP binding site of the gyrase B subunit. In the group of four tested catechins, epigallocatechin gallate (EGCG) had the highest activity, followed by epicatechin gallate (ECG) and epigallocatechin (EGC). Specific binding to the N-terminal 24 kDa fragment of gyrase B was determined by fluorescence spectroscopy and confirmed using heteronuclear two-dimensional NMR spectroscopy of the EGCG-15N-labeled gyrase B fragment complex. Protein residues affected by binding to EGCG were identified through chemical shift perturbation. Molecular docking calculations suggest that the benzopyran ring of EGCG penetrates deeply into the active site while the galloyl moiety anchors it to the cleft through interactions with its hydroxyl groups, which explains the higher activity of EGCG and ECG.

Adenosine Triphosphatases↗