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Abstract Caveolin‐1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin‐1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within theC‐terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full‐lengthS‐alkylated caveolin‐1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI‐LC‐MS analyses verified the addition of the C16 alkyl groups to caveolin‐1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed thatS‐alkylation does not significantly affect theglobalhelicity of the protein; however, molecular dynamics simulations revealed that there werelocalregions where the helicity was altered positively or negatively byS‐alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of theC‐terminal domain, resulting in a disposition within the bilayer characterized by increased depth.