scholarly journals Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis

Nature ◽  
1989 ◽  
Vol 341 (6238) ◽  
pp. 125-130 ◽  
Author(s):  
Joachim Ostermann ◽  
Arthur L. Horwich ◽  
Walter Neupert ◽  
F.-Ulrich Hartl
Keyword(s):  
Protein Folding ◽  
Complex Formation ◽  
Atp Hydrolysis

scholarly journals Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

2021 ◽  
Author(s):  
Thomas E Walker ◽  
Mehdi Shirzadeh ◽  
He Mirabel Sun ◽  
Jacob W McCabe ◽  
Andrew Roth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Cell Function ◽  
Atp Hydrolysis ◽  
Variable Temperature ◽  
Native Mass Spectrometry ◽  
Solution Temperature ◽  
Temperature Dependent ◽  
Chemical Action ◽  
Native Ms

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature-electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL/GroES complexes. The results show clear evidences for de-stabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the pre-viously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry re-veals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiome-try of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temper-ature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the utility of native MS for investiga-tions of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

scholarly journals The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

2013 ◽  
Vol 42 (4) ◽  
pp. 2257-2269 ◽  
Author(s):  
Cecile Evrin ◽  
Alejandra Fernández-Cid ◽  
Alberto Riera ◽  
Juergen Zech ◽  
Pippa Clarke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Complex Formation ◽  
Cell Cycle Arrest ◽  
Atp Hydrolysis ◽  
Cycle Arrest ◽  
Helicase Loading ◽  
Limiting Step ◽  
Prereplicative Complex ◽  
Dominant Lethality

Abstract The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.

scholarly journals Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

2021 ◽  
Author(s):  
Thomas E Walker ◽  
Mehdi Shirzadeh ◽  
He Mirabel Sun ◽  
Jacob W McCabe ◽  
Andrew Roth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Cell Function ◽  
Atp Hydrolysis ◽  
Variable Temperature ◽  
Native Mass Spectrometry ◽  
Solution Temperature ◽  
Temperature Dependent ◽  
Chemical Action ◽  
Native Ms

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature-electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL/GroES complexes. The results show clear evidences for destabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the previously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreport-ed stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J Mol Biol 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the util-ity of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

scholarly journals Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzTPromoter

2016 ◽  
Vol 82 (14) ◽  
pp. 4350-4362 ◽  
Author(s):  
Ana Isabel Platero ◽  
Aroa López-Sánchez ◽  
Laura Tomás-Gallardo ◽  
Eduardo Santero ◽  
Fernando Govantes
Keyword(s):  
Complex Formation ◽  
Promoter Region ◽  
Atp Hydrolysis ◽  
Cyanuric Acid ◽  
Regulatory Protein ◽  
Dna Interaction ◽  
Integration Host Factor ◽  
Open Complex Formation

ABSTRACTPatzTis an internal promoter of theatzRSTUVWoperon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzTis σNdependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety ofin vivoandin vitrogene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT. Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzTpromoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzTpromoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σNand PatzTthat lead to open complex formation.IMPORTANCEHere, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σN-dependent promoter PatzT. Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

scholarly journals The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES

FEBS Letters ◽  
1995 ◽  
Vol 369 (2-3) ◽  
pp. 283-286 ◽  
Author(s):  
Yasushi Kawata ◽  
Kunihiro Hongo ◽  
Koji Nosaka ◽  
Yoshinobu Furutsu ◽  
Tomohiro Mizobata ◽  
...  
Keyword(s):  
Complex Formation ◽  
Atp Hydrolysis ◽  
Chaperonin Groel

scholarly journals Mutant Forms of Salmonella typhimuriumς54 Defective in Transcription Initiation but Not Promoter Binding Activity

1999 ◽  
Vol 181 (11) ◽  
pp. 3351-3357 ◽  
Author(s):  
Mary T. Kelly ◽  
Timothy R. Hoover
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Random Mutagenesis ◽  
Binding Activity ◽  
Mutant Proteins ◽  
Promoter Dna ◽  
Mutant Forms ◽  
Rna Polymerase Holoenzyme

ABSTRACT Transcription initiation with ς54-RNA polymerase holoenzyme (ς54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς54’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium ς54 that were deficient in transcription initiation but still directed ς54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

Incorporation of tubulin subunits into dimers requires GTP hydrolysis

1993 ◽  
Vol 106 (2) ◽  
pp. 627-632 ◽  
Author(s):  
A. Fontalba ◽  
R. Paciucci ◽  
J. Avila ◽  
J.C. Zabala
Keyword(s):  
Protein Folding ◽  
Atp Hydrolysis ◽  
In Vitro Translation ◽  
Magnesium Ions ◽  
Gtp Hydrolysis ◽  
Step Process ◽  
Tubulin Isotypes ◽  
Cytoplasmic Proteins ◽  
Formation Of Complexes

A toroid multisubunit complex of 800–900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different alpha- and beta-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.

Repressor Forms of the Enhancer-binding Protein NtrC: Some Fail in Coupling ATP Hydrolysis to Open Complex Formation by σ54-Holoenzyme

1996 ◽  
Vol 260 (3) ◽  
pp. 317-331 ◽  
Author(s):  
Anne K. North ◽  
David S. Weiss ◽  
Hideyuki Suzuki ◽  
Yehuda Flashner ◽  
Sydney Kustu
Keyword(s):  
Complex Formation ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Enhancer Binding Protein ◽  
Open Complex Formation

scholarly journals Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity

2005 ◽  
Vol 391 (3) ◽  
pp. 481-490 ◽  
Author(s):  
Odile Ramaen ◽  
Christina Sizun ◽  
Olivier Pamlard ◽  
Eric Jacquet ◽  
Jean-Yves Lallemand
Keyword(s):  
Complex Formation ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Activation Mechanism ◽  
Hydrolysis Rate ◽  
Sequential Assignment ◽  
Atp Binding ◽  
Native Page ◽  
Transient Complex

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642–Ser871) and NBD2 (Ser1286–Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10−5 s−1) and NBD2 (6×10−6 s−1) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 μM. Despite the fact that both monomers bind ATP, no stable NBD1·NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1·NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1·NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1·NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

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