The Structure of the Mercury Transporter MerF in Phospholipid Bilayers: A Large Conformational Rearrangement Results from N‑Terminal Truncation

The three-dimensional structure of the 81-residue mercury transporter MerF determined in liquid crystalline phospholipid bilayers under physiological conditions by Rotationally Aligned (RA) solid-state NMR has two long helices, which extend well beyond the bilayer, with a well-defined interhelical loop. Truncation of the N-terminal 12 residues, which are mobile and unstructured when the protein is solubilized in micelles, results in a large structural rearrangement of the protein in bilayers. In the full-length protein, the N-terminal helix is aligned nearly parallel to the membrane normal and forms an extension of the first transmembrane helix. By contrast, this helix adopts a perpendicular orientation in the truncated protein. The close spatial proximity of the two Cys-containing metal binding sites in the three-dimensional structure of full-length MerF provides insights into possible transport mechanisms. These results demonstrate that major changes in protein structure can result from differences in amino acid sequence (e.g., full-length vs truncated proteins) as well as the use of a non-native membrane mimetic environment (e.g., micelles) vs liquid crystalline phospholipid bilayers. They provide further evidence of the importance of studying unmodified membrane proteins in near-native bilayer environments in order to obtain accurate structures that can be related to their functions

Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein–protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities

Tyrosine Replacing Tryptophan as an Anchor in GWALP Peptides

Synthetic model peptides have proven useful for examining fundamental peptide–lipid interactions. A frequently employed peptide design consists of a hydrophobic core of Leu-Ala residues with polar or aromatic amino acids flanking each side at the interfacial positions, which serve to “anchor” a specific transmembrane orientation. For example, WALP family peptides (acetyl-GWW­(LA)_<i>n</i>LWWA-[ethanol]­amide), anchored by four Trp residues, have received particular attention in both experimental and theoretical studies. A recent modification proved successful in reducing the number of Trp anchors to only one near each end of the peptide. The resulting GWALP23 (acetyl-GGALW⁵(LA)₆LW¹⁹LAGA-[ethanol]­amide) displays reduced dynamics and greater sensitivity to lipid–peptide hydrophobic mismatch than traditional WALP peptides. We have further modified GWALP23 to incorporate a single tyrosine, replacing W⁵ with Y⁵. The resulting peptide, Y⁵GWALP23 (acetyl-GGALY⁵(LA)₆LW¹⁹LAGA-amide), has a single Trp residue that is sensitive to fluorescence experiments. By incorporating specific ²H and ¹⁵N labels in the core sequence of Y⁵GWALP23, we were able to use solid-state NMR spectroscopy to examine the peptide orientation in hydrated lipid bilayer membranes. The peptide orients well in membranes and gives well-defined ²H quadrupolar splittings and ¹⁵N/¹H dipolar couplings throughout the core helical sequence between the aromatic residues. The substitution of Y⁵ for W⁵ has remarkably little influence on the tilt or dynamics of GWALP23 in bilayer membranes of the phospholipids DOPC, DMPC, or DLPC. A second analogue of the peptide with one Trp and two Tyr anchors, Y^4,5GWALP23, is generally less responsive to the bilayer thickness and exhibits lower apparent tilt angles with evidence of more extensive dynamics. In general, the peptide behavior with multiple Tyr anchors appears to be quite similar to the situation when multiple Trp anchors are present, as in the original WALP series of model peptides

Properties of Membrane-Incorporated WALP Peptides That Are Anchored on Only One End

Peptides of the “WALP” family, acetyl-GWW­(LA)_<i>n</i>LWWA-[ethanol]­amide, have proven to be opportune models for investigating lipid–peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW­(LA)_<i>n</i>-ethanolamide and acetyl-(AL)_<i>n</i>WWG-ethanolamide, in which <i>n</i> = 4 or 8, which we designate as “N-anchored” and “C-anchored” peptides, respectively. Selected ²H or ¹⁵N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered “half”-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (<i>n</i> = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (<i>n</i> = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of β-structure

Proline Kink Angle Distributions for GWALP23 in Lipid Bilayers of Different Thicknesses

By using selected ²H and ¹⁵N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW⁵LALALALALALALW¹⁹LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) <i>J. Biol. Chem. 285</i>, 31723–31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW⁵LALALAP¹²ALALALW¹⁹LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed ²H and ¹⁵N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined ²H GALA and ¹⁵N–¹H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ∼34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ∼30 ± 5°, with an apparent helix unwinding or “swivel” angle of ∼70°. In DLPC and DOPC, on the basis of ²H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala²¹ in the phospholipids DMPC and DLPC yet remains intact through Ala²¹ in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues