Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.
The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.
Marchenko
N Iu
NIu
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Marchenkov
V V
VV
Kaĭsheva
A L
AL
Kashparov
I A
IA
Kotova
N V
NV
Kaliman
P A
PA
Semisotnov
G V
GV
D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D018833:Chaperonins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification*; D057927:Hydrophobic and Hydrophilic Interactions; D008274:Magnesium / Q000737:chemistry; D009994:Osmolar Concentration; D011489:Protein Denaturation; D055672:Static Electricity
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D018833:Chaperonins; D008274:Magnesium; D002118:Calcium