Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments

Abstract

Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported; all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles. In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences. The helix is weaker between the hydrophobic core and the C-terminus; such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance. No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities. All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity. Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent. The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole. All these comparisons were facilitated by the use of upfield shifts of H alpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.

Cardiolipin modulates the secondary structure of the presequence peptide of cytochrome oxidase subunit IV: a 2D 1H‐NMR study

“…Comparison of the conformation of the peptide in two different micelle systems by comparing the secondary structure-sensitive interresidue NOE cross-peaks was hampered by cross-peak overlap problems and by quantification difficulties due to dynamical effects. The intensities of the medium range NOEs for p25 are relatively low compared with those for the bacterial signal peptides of OmpA [34] or PhoE [35] associated with similar micelles. This can be explained by the fact that p25 is less hydrophobic than the bacterial signal peptides, resulting in a dynamical equilibrium between water-soluble (random coil) and micelle-bound (~-helicab forms of p25.…”

“…It has been shown that the difference between the Ha proton chemical shifts in a protein structure and the chemical shifts in a random coil also correlates with protein secondary structure. An s-helix [34]. For p25 in the presence of DPC micelles, the differences between the Ha chemical shift values with respect to those in water, are given in Fig.…”

Use of synthetic signal sequences to explore the protein export machinery

“…[11][12][13][14] The helix content is highest, and the helix is most stable in the hydrophobic core of the signal sequence. 12,15,16 When the helical conformation required in the targeting pathway was not revealed by these studies, the intrinsic tendency of signal sequences to fold into an a-helix was established.…”

Folding propensities of peptide fragments of myoglobin

“…6), consistent with formation of highly populated helical structure. From the mean C"H shifts, we estimate an average population of 75% helix from Trp 7 to Ala 22 and 57% helix between His 24 and Phe 33 Rizo et al, 1993). From both the C"H chemical shifts and the daN(ir i + 3) NOEs, there appears to be a break in the helix at Ala 22 and Gly 23.…”