GroEL-GroES Cycling

112

158

Recent experiments suggest that the folding of certain proteins can take place entirely within a chaperonin-like cavity. These substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. Rather, they undergo only a single binding event, followed by sequestration into the chaperonin cage. The present work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within this chaperonin cavity. The chaperonin interior is modeled by a sphere with a lining of tunable degree of hydrophobicity. We demonstrate that a moderately hydrophobic environment, similar to the interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations support a mechanism by which the moderately hydrophobic chaperonin environment provides an alternate pathway to the native state through a transiently bound intermediate state.

Section: Discussionmentioning

confidence: 99%

Accelerated folding in the weak hydrophobic environment of a chaperonin cavity: Creation of an alternate fast folding pathway

Jewett

Baumketner

Shea

2004

112

158

“…Purified complexes were dialyzed against 50 mM Tris, pH 7.5͞50 mM KCl͞1 mM tris(carboxyethyl)phosphine before labeling. GroES Cys98 was produced as described (18). Pig heart mitochondrial malate dehydrogenase and hexokinase were purchased from Roche Applied Science.…”

Section: Methodsmentioning

confidence: 99%

“…ATP hydrolysis, by contrast, is used to directionally advance the machine from its folding-active state toward the binding-active state. Notably, the longest part of the ATP-driven chaperonin reaction cycle (t 1/2 Ϸ 10 sec) is the folding-active ATP-bound (and cochaperonin-bound) state (18). The hydrolysis timer appears to be slow enough to optimize the ability of proteins to fold in the enclosed cavity, but it also has to be sufficiently fast to release polypeptides from the chaperonin, allowing them, for example, to assemble with partners into oligomeric protein, but more generally to be accessible in the cell to carry out work.…”

mentioning

confidence: 99%

Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL

Motojima

Chaudhry

Fenton

et al. 2004

Self Cite

A conundrum has arisen in the study of the structural states of the GroEL-GroES chaperonin machine: When either ATP or ADP is added along with GroES to GroEL, the same asymmetric complex, with one ring in a GroES-domed state, is observed by either x-ray crystallographic study or cryoelectron microscopy. Yet only ATP͞ GroES can trigger productive folding inside the GroES-encapsulated cis cavity by ejecting bound polypeptide from hydrophobic apical binding sites during attendant rigid body elevation and twisting of these domains. Here, we show that this difference occurs because polypeptide substrate in fact presents a load on the apical domains, and, although ATP can counter this load effectively, ADP cannot. We monitored apical domain movement in real time by fluorescence resonance energy transfer (FRET) between a fixed equatorial fluorophore and one attached to the mobile apical domain. In the absence of bound polypeptide, addition of either ATP͞GroES or ADP͞GroES to GroEL produced the same rapid rate and extent of decrease of FRET (t 1/2 < 1 sec), reflecting similarly rapid apical movement to the same end-state and explaining the results of the structural studies, which were all carried out in the absence of substrate polypeptide. But in the presence of bound malate dehydrogenase or rhodanese, whereas similar rapid and extensive FRET changes were observed with ATP͞GroES, the rate of FRET change with ADP͞GroES was slowed by >100-fold and the extent of change was reduced, indicating that the apical domains opened in a slow and partial fashion. These results indicate that the free energy of ␥-phosphate binding, measured earlier as 43 kcal per mol (1 cal ‫؍‬ 4.184 J) of rings, is required for driving the forceful excursion or ''power stroke'' of the apical domains needed to trigger release of the polypeptide load into the central cavity.

“…Hsps are essential for cell survival in all species subjected to stress. Hsps have a chaperone function, demonstrated by their ability to bind proteins and mediate conformational changes during protein folding (13).…”

mentioning

confidence: 99%

Heat-shock protein 60 is required for blastema formation and maintenance during regeneration

Makino

Whitehead

Lien

et al. 2005

Zebrafish fin regeneration requires the formation and maintenance of blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for blastema formation and maintenance. We used a chemical mutagenesis screen to identify no blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue.blastema ͉ regeneration ͉ zebrafish ͉ genetics ͉ stress response