Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins.
Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.
Schoeffler
Allyn J
AJ
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Ruiz
Carmen R
CR
Joubert
Allison M
AM
Yang
Xuemei
X
LiCata
Vince J
VJ
D017667:Adipocytes / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D050356:Lipid Metabolism; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D011189:Potassium Chloride / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012492:Salts / Q000494:pharmacology*; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008024:Ligands; D012492:Salts; C027132:1-anilino-8-naphthalenesulfonate; D011189:Potassium Chloride