scholarly journals Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability Using Mass Spectrometry

2012 ◽  
Vol 84 (3) ◽  
pp. 1653-1660 ◽  
Author(s):  
Duc T. Tran ◽  
Sambuddha Banerjee ◽  
Abdu I. Alayash ◽  
Alvin L. Crumbliss ◽  
Michael C. Fitzgerald
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Thermodynamic Analysis

Investigation of protein folding by mass spectrometry

1996 ◽  
Vol 10 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Andrew Miranker ◽  
Carol V. Robinson ◽  
Sheena E. Radford ◽  
Christopher M. Dobson
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding

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 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.

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