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Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix.

ABSTRACT: Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function. The disorder can involve molecular orientation as well as molecular folding. This paper reports an astonishing induction of disorder when one Glu residue is introduced into a highly stable 23-residue transmembrane helix. The parent helix is anchored by a single Arg residue, tilted at a well-defined angle with respect to the DOPC bilayer normal and undergoes rapid cone precession. When Glu is introduced two residues away from Arg, with 200° (or 160°) radial separation, the helix properties change radically to exhibit a multiplicity of three or more disordered states. The helix characteristics have been monitored by deuterium (²H) NMR spectroscopy as functions of the pH and lipid bilayer composition. The disordered multistate behavior of the (Glu, Arg)-containing helix varies with the lipid bilayer thickness and pH. The results highlight a fundamental induction of protein multistate properties by a single Glu residue in a lipid membrane environment.

SUBMITTER: Price JR

PROVIDER: S-EPMC8359125 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59]. We have further investigated this transition and find that the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterioopsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are 5.46 for residues 72-248 (Cys 163) and 7.36 for residues 1-166 (Lys 41). When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding in the range of -0.54 to -3.5 kcal/mol. The results suggest that the C-terminal helix of bacterioopsin is less stably folded than the N-terminal helices. We analyzed the pairwise helix-helix interaction surfaces of bacteriorhodopsin and three other seven-transmembrane-helix proteins on the basis of crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl-terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high-resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix interactions.

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Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix.

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