The sterol carrier protein-2 fatty acid binding site: an NMR, circular dichroic, and fluorescence spectroscopic determination.
The interaction and orientation of fatty acids with recombinant human sterol carrier protein-2 (SCP-2) were examined by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence techniques. 13C-NMR spectroscopy of stearic acid and oleic acid as well as fluorescence spectroscopy of cis-parinaric acid demonstrated that SCP-2 bound naturally occurring fatty acids with near 1:1 stoichiometry. Several findings indicated that the fatty acid was oriented in the binding site with its methyl end buried in the protein interior and its carboxylate exposed at the surface: the chemical shift of bound [18-13C]-stearate; dicarboxylic/monocarboxylic acid cis-parinaric acid displacement; complete ionization of the carboxylate group of SCP-2 bound [1-13C]stearate at neutral pH; lack of electrostatic interactions between 13C-fatty acids with SCP-2 cationic residues: pH titratability of the SCP-2 bound [1-13C]stearate carboxylate group. SCP-2 did not undergo global structural changes upon ligand binding or pH decrease as indicated by the absence of significant changes in NMR and only small alterations in time resolved fluorescence parameters. However, SCP-2 did undergo secondary structural changes detected by CD in the pH range 5-6. While these changes in secondary structure did not alter the fatty acid:SCP-2 binding stoichiometry, the affinity for fatty acid was increased severalfold at lower pH. In summary, 13C-NMR, CD, and fluorescence spectroscopy provided a detailed understanding of the interaction of fatty acids with SCP-2 and further showed for the first time the orientation of the fatty acid within the binding site. The pH-induced changes in SCP-2 secondary structure and ligand binding activity may be important to the mechanism whereby this protein interacts with membrane surfaces to enhance lipid binding/transfer.
Stolowich
N J
NJ
Department of Chemistry, Texas A&M University, College Station 77843-3255, USA.
Frolov
A
A
Atshaves
B
B
Murphy
E J
EJ
Jolly
C A
CA
Billheimer
J T
JT
Scott
A I
AI
Schroeder
F
F
D001665:Binding Sites; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010940:Plant Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013261:Sterols / Q000378:metabolism*
D002247:Carbon Isotopes; D002352:Carrier Proteins; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D005231:Fatty Acids, Unsaturated; D010940:Plant Proteins; D011994:Recombinant Proteins; D013261:Sterols; C072087:sterol carrier proteins; C013641:parinaric acid