Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structur...

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Bibliographic Details
Published in:Biochemistry (Easton) 2008-06, Vol.47 (22), p.5915-5934
Main Authors: Martin, Gregory G, Hostetler, Heather A, McIntosh, Avery L, Tichy, Shane E, Williams, Brad J, Russell, David H, Berg, Jeremy M, Spencer, Thomas A, Ball, Judith, Kier, Ann B, Schroeder, Friedhelm
Format: Article
Language:English
Subjects:
Binding Sites
Carrier Proteins - chemistry
Carrier Proteins - metabolism
Fluorescence Resonance Energy Transfer
Humans
Ligands
Protein Precursors - chemistry
Protein Precursors - metabolism
Protein Structure, Secondary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Spectrometry, Fluorescence
Structure-Activity Relationship
Tryptophan - chemistry
Tryptophan - metabolism
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Summary:Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi800251e