pH and cation-induced thermodynamic stability of human hyaluronan binding protein 1 regulates its hyaluronan affinity.
Hyaluronan-binding protein 1 (HABP1) is a trimeric protein with high negative charges distributed asymmetrically along the faces of the molecule. Recently, we have reported that HABP1 exhibits a high degree of structural flexibility, which can be perturbed by ions under in vitro conditions near physiological pH (Jha, B. K., Salunke, D. M., and Datta, K. (2003) J. Biol. Chem. 278, 27464-27472). Here, we report the effect of ionic strength and pH on thermodynamic stability of HABP1. Trimeric HABP1 was shown to unfold reversibly upon dissociation ruling out the possibility of existence of folded monomer. An increase in ionic concentration (0.05-1 M) or decrease in pH (pH 8.0-pH 5.0) induced an unusually high thermodynamic stability of HABP1 as reflected in the gradual increase in transition midpoint temperature, enthalpy of transition, and conformational entropy. Our studies suggest that the presence of counter ions in the molecular environment of HABP1 leads to dramatic reduction of the intramolecular electrostatic repulsion either by de-ionizing the charged amino acid residues or by direct binding leading to a more stable conformation. A regulation on cellular HA-HABP1 interaction by changes in pH and ionic strength may exist, because the more stable conformation attained at higher ionic strength or at acidic pH showed maximum affinity toward HA as probed either in solid phase binding assay on HA-immobilized plates or an in-solution binding assay using intrinsic fluorescence of HABP1.
Jha
Babal Kant
BK
103 Biochemistry Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
Mitra
Nivedita
N
Rana
Rachita
R
Surolia
Avadhesha
A
Salunke
Dinakar M
DM
Datta
Kasturi
K
D002352:Carrier Proteins; D002412:Cations; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D024101:Mitochondrial Proteins; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
C087299:C1QBP protein, human; D002352:Carrier Proteins; D002412:Cations; D018960:Hyaluronan Receptors; D024101:Mitochondrial Proteins; D006820:Hyaluronic Acid