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J. Biol. Chem., Vol. 280, Issue 3, 2045-2054, January 21, 2005

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pH-dependent Interactions of the Carboxyl-terminal Helix of Steroidogenic Acute Regulatory Protein with Synthetic Membranes^*

Dustin C. Yaworsky¶, Bo Y. Baker, Himangshu S. Bose||, Katrina B. Best**, Lauren B. Jensen**, John D. Bell**, Michael A. Baldwin, and Walter L. Miller

From the Department of Pediatrics and the Metabolic Research Unit and the Department of Pharmaceutical Chemistry Mass Spectrometry Facility, University of California San Francisco, San Francisco, California 94143 and the ^**Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah 84602

Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259–282; at pH 6.5 this region was partially digested into 254–272, 254–273, and 254–274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal 4 helix and that residues Leu²⁷⁵, Thr²⁶³, and Arg²⁷² in 4 form stabilizing interactions with Gln¹²⁸, Asp¹⁵⁰, and Asp¹⁰⁶ in adjacent loops. CD spectroscopy of a 37-mer model of 4 (residues 247–287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was -helical and achieved maximal -helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal 4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the -helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane.

Received for publication, September 23, 2004 , and in revised form, October 14, 2004.

^* This work was supported by NIH Grant DK37922 (to W. L. M.) and by National Science Foundation (NSF) Grant MCB 9904597 (to J. D. B.). The mass spectrometry was performed at the UCSF Mass Spectrometry Facility supported by NIH Grant RR01614. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported in part by Pediatric Endocrinology Training Grant DK07161 (to W. L. M.).

^|| Supported by NIH Grant KO1 DK02762. Present address: Dept. of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610.

To whom correspondence should be addressed: University of California San Francisco, Bldg. MR IV, Rm. 205, Box 0978, San Francisco, CA 94143-0978. E-mail: wlmlab{at}itsa.ucsf.edu.

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