HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

Abstract

Membrane protein Nramp1 (natural resistance-associated macrophage protein 1) is a pH-dependent divalent metal cation transporter that regulates macrophage activation in infectious and autoimmune diseases. A naturally occurring glycine to aspartic acid substitution at position 169 (G169D) within the transmembrane domain 4 (TM4) of Nramp1 makes mice susceptible to Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis. Here we present a structural and self-assembling study on two synthetic 24-residue peptides, corresponding to TM4 of mouse Nramp1 and its G169D mutant, respectively, in 1,1,1,3,3,3-hexafluoroisopropanol-d(2) (HFIP-d(2)) aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. The results show that amphipathic alpha-helical structures are formed from residue Ile173 to Tyr187 for the wild-type peptide and from Trp168 to Tyr187 for the G169D mutant, respectively. The segment of the N-terminus from Leu167 to Leu172 is poorly structured for the wild-type peptide, whereas it is well defined for the G169D mutant. Both peptides aggregate to form a tetramer and the monomeric peptides in peptide bundles are structurally and orientationally similar. The intermolecular interactions in assemblies could be stronger in the C-terminal regions related to residues Phe180-Leu184 than those in the central helical segments for both peptides. The G169D mutation may change the size of the opening on the termini of assembly.

Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

“…In contrast, the molar ellipticity of the R9AP-Cter peptide is much smaller in HFIP than in HMeOH (Fig. 2G), which is not consistent with previous observations that this solvent favors the formation of α-helical structures (see section 3.1) [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103].…”

“…Nevertheless, care must be taken when using TFE and HFIP because it is generally believed that they enhance the population of helical conformations of peptides and proteins. In particular, since the pioneering work of Tamburro et al [88], fluorinated alcohols, such as TFE and HFIP, have been shown to promote the formation of α-helices for a large number of proteins and peptides (see, for example, references [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] and the references cited therein). It has been proposed that fluoroalcohol complexes would displace water molecules from the surface of the peptide; the acidity or hydrogen-bonding ability and hydrophobicity of the fluoroalcohols appear to play an important role [104].…”

“…2F does not fit well with any of the infrared spectra of this peptide; indeed, the spectrum in HFIP indicates a long and rigid α-helix whereas that in H-MeOH and the powder respectively reveal a large content in turns and β-sheets. This raises also the issue that HFIP could favor the formation of α-helical structures in the case of this peptide [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103].…”

α-Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins

“…Thus, while organic solvents are frequently used in structural studies on single, monomeric peptides, they are widely considered unsuitable for investigations of helix-helix interactions and peptide oligomerization. Using diffusion-ordered spectroscopy (DOSY) NMR, however, Sun and coworkers discovered peptide oligomers even in 100% TFE Xue et al, 2008Xue et al, , 2009 or 40% HFIP (Li et al, ,c, 2008Xue et al, 2006). The authors determined high-resolution structures of oligomers composed of peptides corresponding to TM helix 4 of solute carrier family 11 member 2 (Slc11a2, formerly known as divalent metal transporter 1 (DMT1)) and TM helix 4 of the homologous protein Slc11a1 (formerly known as natural resistance-associated macrophage protein 1 (Nramp1)).…”

“…Even if more convincing evidence of peptide oligomers in SDS micelles can be found, it still remains to be shown that they possess pore activity. To settle the uncertainties surrounding Slc11a2 TM helix 4, and a similar case involving TM helix 4 of Slc11a1 (Xue et al, 2006(Xue et al, , 2008(Xue et al, , 2009, further experiments are urgently needed.…”