Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay.
Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.
Stanley
Will A
WA
EMBL-Hamburg, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany. stanley@embl-hamburg.de
Versluis
Kees
K
Schultz
Carsten
C
Heck
Albert J R
AJ
Wilmanns
Matthias
M
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002331:Carnitine / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D008024:Ligands; C072087:sterol carrier proteins; D002331:Carnitine