Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH.
Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH(cal)/ΔH(van) approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.
School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Prasanna K
PK
Devaraneni
Neeraj
N
Mishra
Rajiv
R
Bhat
D002942:Circular Dichroism; D019855:Ethylene Glycol / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006593:Hexokinase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011108:Polymers / Q000737:chemistry*; D011489:Protein Denaturation; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013012:Sorbitol / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology*
D011108:Polymers; D012997:Solvents; C024617:polyol; D013012:Sorbitol; D014508:Urea; D006593:Hexokinase; D019855:Ethylene Glycol; D005947:Glucose; D005990:Glycerol