In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with...

The molecular chaperones GroEL and GroES were produced at very high levels in Escherichia coli, purified, and shown to protect pig mitochondrial malate dehydrogenase (MDH) against thermal inactivation in vitro. The apparent rate of MDH inactivation at 37 degrees C was reduced by a factor of at least 5 in a process which required only GroEL, GroES, and ATP. GroEL alone did not protect MDH agains...

BACKGROUND The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit. METHODOLOGY/PRINCIPAL FINDINGS Three soluble, partially active mutants with polypepti...

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shape...

Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 m urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg2+ complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small ...

We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then ...

Escherichia coli chaperonin, GroEL, helps proteins fold under nonpermissive conditions. During the reaction cycle, GroEL undergoes allosteric transitions in response to binding of a substrate protein (SP), ATP, and the cochaperonin GroES. Using coarse-grained representations of the GroEL and GroES structures, we explore the link between allosteric transitions and the folding of a model SP, a de...

The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cle...

Background: Chaperonins like the GroEL-GroES complex facilitate protein folding in the cell. Results: Substrate proteins are captured by the open, trans ring of the GroEL-ATP-GroES complex and are partially unfolded. Conclusion: Maximally efficient folding requires repeated cycles of substrate protein unfolding by the GroEL-GroES complex. Significance: Establishing how substrate proteins are pr...

The nonhydrolyzable ATP analogue ATP gamma S (adenosine 5'-3-O-(thio)triphosphate) is affinity cross-linked to GroEL by formation of a disulfide bridge in a peroxide-promoted reaction. By replacing with serine each of 3 cysteine residues in GroEL, it is shown that ATP gamma S specifically cross-links to Cys-137. It is thus demonstrated that the ATP bound to GroEL is in direct contact with Cys-137.