NMR determination of the secondary structure and the three‐dimensional polypeptide backbone fold of the human sterol carrier protein 2

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Abstract
Nuclear magnetic resonance (NMR) spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of the human sterol carrier protein 2 (hSCP2), which is a basic protein with 123 residues believed to participate in the intracellular transport of cholesterol and various other lipids. Sequence-specific assignments were obtained for nearly all backbone 1H and 15N resonances, as well as for about two-thirds of the side-chain 1H resonances, using uniform15N-labeling of the protein combined with homonuclear two-dimensional1H NMR and three-dimensional 15N-correlated 1H NMR. Three α-helices comprising the polypeptide segments of residues 9–22, 25–30 and 78–84 were identified by sequential and medium-range nuclear Overhauser effects (NOE). The analysis of long-range backbone-backbone NOEs showed that hSCP2 further contains a five-stranded β-sheet including the residues 33-41, 47-54, 60-62, 71-76 and 100-102, which is a central feature of the molecular architecture. The first three strands are arranged in an antiparallel fashion, the polypeptide chain then crosses over this three-stranded sheet in a right-handed sense so that the fourth strand is added parallel to the first one. The fifth strand runs antiparallel to the fourth one, so that the overall topology is +1, +1, −3x, −1. The three-dimensional arrangement of the β-sheet and the first two helices was determined using an input of 625 NOE upper distance constraints and 95 scalar coupling constants for a preliminary structure calculation with the distance geometry program DIANA.

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