Hydrophobic core of the steroidogenic acute regulatory protein for cholesterol transport.
The steroidogenic acute regulatory protein (StAR), the first family member of START (StAR-related lipid transport) proteins, plays an essential role by facilitating the movement of cholesterol from the outer to inner mitochondrial membrane. Wild-type and mutant StAR binds cholesterol with similar intensity, but only wild-type StAR can transport it to mitochondria. Here, we report that the hydrophobic core is crucial for biological activity of proteins with START domains. Wild-type StAR increased steroidogenic activity by 7-9-fold compared to mutant R182L StAR, but both of them showed similar near-UV CD spectra. The fluorescence maximum of wild-type StAR is red shifted in comparison to mutant StAR under identical urea concentration. TFE increased the alpha-helical contribution of wild-type StAR more than the mutant protein. Acrylamide quenching for the wild-type protein (K(SV) = 12.0 +/- 0.2-11.2 +/- 0.5 M(-1)) exceeded that of the mutant protein (K(SV) = 4 +/- 0.2 M(-1)). Consistent with these findings, the hydrophobic probe ANS bound wild-type StAR (K(app) = 8.1 x 10(5) M(-1)) to a greater degree than mutant StAR (K(app) = 3.75 x 10(5) M(-1)). Partial proteolysis examined by mass spectrometry suggests that only wild-type StAR has a protease-sensitive C-terminus, but not the mutant. Stopped-flow CD revealed that the time of unfolding of mutant StAR was 0.017 s. In contrast, the wild-type StAR protein is unfolded in 16.3 s. In summary, these results demonstrate that wild-type StAR adopts a very flexible form due to the accommodation of more water molecules, while mutant StAR is generated by an alternate folding pathway making it inactive.
Bose
Himangshu S
HS
Department of Biomedical Sciences, Mercer University School of Medicine and Memorial Health University Medical Center, Savannah, Georgia 31404, USA. bose_hs@mercer.edu
Whittal
Randy M
RM
Bose
Mahuya
M
Debnath
Dilip
D
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism*; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D018583:Pliability / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D034421:Sus scrofa; D014270:Trifluoroethanol / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D000098682:Steroidogenic Acute Regulatory Protein
D000817:Anilino Naphthalenesulfonates; D050505:Mutant Proteins; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D014364:Tryptophan; D014508:Urea; D002784:Cholesterol