TY - JOUR
T1 - Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH
AU - Devaraneni, Prasanna K.
AU - Mishra, Neeraj
AU - Bhat, Rajiv
PY - 2012/4
Y1 - 2012/4
N2 - 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.
AB - 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.
KW - Calorimetry
KW - Cooperative structure
KW - Molten globule
KW - Osmolytes
KW - Polyols
KW - Thermal stability
KW - Yeast hexokinase A
UR - http://www.scopus.com/inward/record.url?scp=84857453944&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84857453944&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2011.12.012
DO - 10.1016/j.biochi.2011.12.012
M3 - Article
C2 - 22203099
AN - SCOPUS:84857453944
SN - 0300-9084
VL - 94
SP - 947
EP - 952
JO - Biochimie
JF - Biochimie
IS - 4
ER -