Solid-State NMR Studies of a Diverged Microsomal Amino-Proximate ¹² Desaturase Peptide Reveal Causes of Stability in Membrane: Tyrosine Anchoring and Arginine Snorkeling

¹ Miami University

^* To whom correspondence should be addressed. E-mail: lorigag{at}muohio.edu.

Submitted on June 2, 2005
Revised on July 20, 2005
Accepted on 28 October 2005

	Abstract

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal ¹²-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with ²H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50) and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol% peptide to lipid, ³¹P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the Lphase. The ²H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz indicating increased side chain motion and solvent-exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the ²H-labelled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8° with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, the conformation in a biomembrane mimic, and presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases and a model of arginine and tyrosine control of transmembrane protein stability and insertion.

Key Words: acetylenase, membrane protein, phospholipid bilaye, solid-state NMR