Bilayer thickness determines the alignment of model polyproline helices in lipid membranes

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.DFG, 207100805, FOR 1805: Einfluss der Ribosomendynamik auf Regulation der Geschwindigkeit und Genauigkeit der TranslationTU Berlin, Open-Access-Mittel - 201

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides—A Solid-State 19F NMR Study

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A $^{19}$F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state $^{19}$F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes

Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a typical thinning response in the case of PGLa, and the increase in the repeat distance and membrane thickening observed for TisB, demonstrate that the concept of peptide-induced membrane thinning cannot be generalized. Instead, these results suggest that different factors contribute to the resulting changes in membrane thickness, such as the peptide orientation in the bilayer, and/or bilayer adaptation to hydrophobic mismatch

Transformation of the matrix structure of shrimp shells during bacterial deproteination and demineralization

BACKGROUND: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and (13)C solid state NMR spectroscopy. RESULTS: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.(13)C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product. CONCLUSIONS: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19^{19}F‐MRI

A labeling strategy for in vivo $^{19}$F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and β-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 $^{19}$F substitutions chemically equivalent, giving rise to a single $^{19}$F-NMR resonance with <10 Hz linewidth. The resulting performance in $^{19}$F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in $^{19}$F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo $^{19}$F-MRI applications

Folding and Self-Assembly of the TatA Translocation Pore Based on a Charge Zipper Mechanism

SummaryWe propose a concept for the folding and self-assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other membrane proteins based on electrostatic “charge zippers.” Each subunit of TatA consists of a transmembrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complementary to the charge pattern on the APH, suggesting that the protein can be “zipped up” by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a transmembrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by monitoring the monomer-oligomer equilibrium of specific charge mutants. Similar “charge zippers” are proposed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral ERNS protein

Identifying Anisotropic Constraints in Multiply Labeled Bacteriorhodopsin by (15)N MAOSS NMR: A General Approach to Structural Studies of Membrane Proteins

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically (15)N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the (15)N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results