Two‐Dimensional ¹H‐NMR of Transmembrane Peptides from Escherichia Coli Phosphatidylglycerophosphate Synthase in Micelles

Abstract

Two 28-residue peptides, PTLLTLFRVILIPFFVLVFYKKKGKKKG [Pgs-(6-25)-peptidyl-KKKGKKKG; Pgs peptide A] and VEYAGIALFFVAAVLTLWSMLQYLSAAR )-peptide, Pgs peptide El, were synthesized and studied by CD and two-dimensional 'H-NMR spectroscopy. The first 20 amino acid residues of Pgs peptide A are identical to one predicted transmembrane segment (Pro6 -Tyr25) of the integral membrane protein phosphatidylglycerophosphate synthase (Pgs) of Escherichiu coli. Pgs peptide E is identical to another predicted transmembrane segment (Va1149-Arg176), which is located in the C-terminal end of this lipid synthase. Pgs peptides A and E were dissolved in methanol or trifluoroethanol or were incorporated into solvent-free micelles of fully deuterated SDS. In all these systems, CD spectra of both peptides indicated an a-helical secondary structure. However, peptides that were solubilized in micelles exhibited the highest content of a-helix as judged from comparison of the CD spectra. Thermodynamically stable isotropic solutions at high peptide concentrations (1 -3 mM) could only be obtained with the peptide incorporated in micelles; in organic solvents, significant peptide aggregation occurred. Relatively sharp peaks were obtained with 'H-NMR spectroscopy of the peptides in SDS micelles, which indicates rapid tumbling of the peptides in the micellar environment. Translational-diffusion coefficients of the micelles with and without peptide, determined by pulsed-fieldgradient NMR, showed that the micellar size was unaffected by the solubilized peptide. The radius of the hydrated micelles was estimated to be about 2.7 nm (i.e. the mass of the aggregate is almost 30 kDa). Two-dimensional NMR spectroscopy of both peptides solubilized in the micelles indicated an a-helical conformation. This observation is strengthened by an investigation of the hydrogen exchange of the peptide amide protons, where significantly less exchange of the amide protons was observed in the middle of the peptides compared with the ends.

Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide

“…Similar results were obtained for several membrane-surfacebinding peptides, including melittin in DPC [6], opioid peptides bound to DPC [30], and a fragment of human apolipoprotein C-I in complex with SDS [31]. In addition, two transmembrane peptides were also shown to have little eVect on the size of the SDS micelle [32]. It appears to be general that the micelles dominate the diVusion rate of the peptide/micelle complexes irrespective of the type of peptides.…”

Determination of the Binding Constant of a Protein Kinase C Substrate, NG(28–43), to Sodium Dodecyl Sulfate via the Diffusion Coefficient Measured by Pulsed Field Gradient Nuclear Magnetic Resonance

“…study at 298 K. In 52 mM SDS micellar solution, the average value of the measured diffusion coefficient is 9.2 ϫ 10 Ϫ7 cm 2 /s. This is close to the value of 8.6 ϫ 10 Ϫ7 cm 2 /s for the SDS micelle based on a previous measurement (26). (28 -43) to the SDS Micelle Upon introduction of the SDS micelle to the peptide solution, a substantial reduction in the diffusion coefficient for the peptide is observed.…”

“…The interactions between the melittin:micelle complex (27), the dynorphin A:micelle complex (30), and the phosphatidylglycerophosphate synthase (Pgs) peptide of Escherichia coli in micellar solution (26) have also been investigated by D s measurements. Results from these studies indicated that, within the experimental error, the micelles are of the same size in the presence and absence of bound protein/peptide.…”

“…At 298 K, the critical micelle concentration (CMC) for SDS is 8 mM with an aggregation number of 62 (25). In the two-state fast-exchange approximation, the observed diffusion coefficient (D s,obs ) of NG (28 -43) in the presence of the micelle is the population-weighted average of the values of free species (D s,f ) and the bound species (D s,b ) (26,27).…”

Comparative genomic analysis of the flagellin glycosylation island of the Gram-positive thermophile Geobacillus

“…2.7.7.39), while Pgs catalyzes the conversion of CDP-diacylglycerol to phosphatidylglycerophosphate (E.C. 2.7.8.5) [53, 54]. The presence of these two key enzymes of phospholipid biosynthesis suggests that the flagellin in this strain may be modified with a phospholipid derivative.…”