Peptide Length Research Articles

Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor.

Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.

In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation.

Analysis of protein C termini is very important for functional annotations of proteomes, while proteome-wide C termini analysis still poses substantial challenges. Here we described a simple and robust strategy for specific isolation of protein C termini based on LysC digestion and site-selective dimethylation to deplete N-terminal and internal peptides by scavenger materials. The performance of LysC digestion and conditions of site-selective dimethylation and resin coupling were discussed in detail. Then the strategy was successfully applied to the characterization of protein C termini of HeLa cells. A total of 781 protein C termini were identified with a 300 μg digest in our study, among which 38.9% were actually not identifiable using current trypsin digestion-based methods due to their inappropriate peptide length for MS analysis, indicating that our method was highly complementary to the existing methods. The enrichment procedure was rapid and easy to operate and could afford a very good identification efficiency by obtaining the largest C termini data set of the human proteome with the least sample loading. This method was without bias toward physicochemical properties of peptides. Moreover, a peptide-centric database was first introduced to analyze protein C termini, which effectively improved the accuracy and speed of the database search. Therefore, our method can be used to effectively and selectively isolate protein C termini and contributes to the global annotation of C terminomes.

Twisted Ribbon Aggregates in a Model Peptide System.

The model peptides A8K and A10K self-assemble in water into ca. 100 nm long ribbon-like aggregates. These structures can be described as β-sheets laminated into a ribbon structure with a constant elliptical cross-section of 4 by 8 nm, where the longer axis corresponds to a finite number, N ≈ 15, of laminated sheets, and 4 nm corresponds to a stretched peptide length. The ribbon cross-section is strikingly constant and independent of the peptide concentration. High-contrast transmission electron microscopy shows that the ribbons are twisted with a pitch λ ≈ 15 nm. The self-assembly is analyzed within a simple model taking into account the interfacial free energy of the hydrophobic β-sheets and a free energy penalty arising from an increased stretching of hydrogen bonds within the laminated β-sheets, arising from the twist of the ribbons. The model predicts an optimal value N, in agreement with the experimental observations.

In Silico Peptide Ligation: Iterative Residue Docking and Linking as a New Approach to Predict Protein-Peptide Interactions.

Peptide–protein interactions are corner-stones of living functions involved in essential mechanisms, such as cell signaling. Given the difficulty of obtaining direct experimental structural biology data, prediction of those interactions is of crucial interest for the rational development of new drugs, notably to fight diseases, such as cancer or Alzheimer’s disease. Because of the high flexibility of natural unconstrained linear peptides, prediction of their binding mode in a protein cavity remains challenging. Several theoretical approaches have been developed in the last decade to address this issue. Nevertheless, improvements are needed, such as the conformation prediction of peptide side-chains, which are dependent on peptide length and flexibility. Here, we present a novel in silico method, Iterative Residue Docking and Linking (IRDL), to efficiently predict peptide–protein interactions. In order to reduce the conformational space, this innovative method splits peptides into several short segments. Then, it uses the performance of intramolecular covalent docking to rebuild, sequentially, the complete peptide in the active site of its protein target. Once the peptide is constructed, a rescoring step is applied in order to correctly rank all IRDL solutions. Applied on a set of 11 crystallized peptide–protein complexes, the IRDL method shows promising results, since it is able to retrieve experimental binding conformations with a Root Mean Square Deviation (RMSD) below 2 Å in the top five ranked solutions. For some complexes, IRDL method outperforms two other docking protocols evaluated in this study. Hence, IRDL is a new tool that could be used in drug design projects to predict peptide–protein interactions.

The Effect of Substrate Length on Lysine Deacetylase Activity

Lysine deacetylases (KDACs) are proteins involved in regulating post‐translational modification of lysine residues in proteins. Anomalous acetylation of lysine has been linked to numerous diseases, and unusual activity of lysine deacetylases often appears to be a causative factor. Our goal is to understand the substrate specificity of metal‐dependent KDACs. Here we are specifically interested in the correlation between KDAC activity with peptides derived from acetylated proteins and the activity of the same KDAC with the corresponding full‐length substrate proteins. Activity with peptides is determined by both label‐free fluorescence assays and mass spectrometry. Initial results with peptides, each derived from a single source protein but of varying length of sequence on either side of the acetylated lysine, did not reveal a correlation between peptide length and KDAC activity. The absence of such a correlation suggests important contributions from secondary structure of the substrate protein and/or contributions from amino acid residues distant from the acetylated lysine in sequence. Ongoing work with full‐length substrate proteins, obtained through recombinant expression with a site‐specific acetyllysine or using immunoprecipitation from human cell lines, provides further insight into substrate features contributing to KDAC activity. Correlation, or the lack thereof, of the activity of the KDACs with the peptide models and full‐length substrates serves as an important control on models predicting KDAC activity, which are typically based on peptides of limited variability. Understanding how substrate size and structure affect the overall activity of KDACs will also provide insight into their substrate specificity and biological targets.Support or Funding InformationThe Louisiana Cancer Research Consortium, National Institutes of Health 5G12MD007595 and TL4GM118968, National Science Foundation CHE 1625993 and MCB 1817358, and the U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A short peptide potentially promotes the healing of skin wound.

Skin wound, a common form of skin damage in daily life, remains a serious challenge in clinical treatment. Bioactive peptides with high efficiency have been considered as potential therapeutic candidates for wound healing. In this report, a novel short linear peptide, with mature peptide sequence of ‘GLLSGINAEWPC’ and no obvious similarity with other known bioactive peptides, was identified by genomic method from the skin of odorous frog, Odorrana andersonii. Our results suggested that OA-GL12 (OA: abbreviation of species (O. andersonii), GL: two initial amino acids, 12: peptide length) obviously accelerated the scratch-healing of keratinocytes and human fibroblasts in a time- and concentration-dependent manner. Meanwhile, OA-GL12 showed significant effect in promoting the wound healing on the full-thickness skin wound model. Inflammatory assay results demonstrated that OA-GL12 induced the secretion of tumor necrosis factor (TNF) and transforming growth factor β1 (TGF-β1) on murine macrophage cell line (RAW264.7), which might explain the powerful accelerating capacity of wound healing. Moreover, results also indicated that epidermal growth factor receptor (EGFR) was involved in the mechanisms underlying the scratch-healing promoting activity of OA-GL12. In addition, OA-GL12 showed obvious free radical scavenging activity. Results supported that OA-GL12 did not exert risk in acute toxicity, hemolytic activity, and direct antibacterial activity. The remarkable effect of OA-GL12 on promoting wound healing verified in this research made it potential to be a novel template for the development of wound healing-promoting agents.

Localization of 99mTc-GRP Analogs in GRPR-Expressing Tumors: Effects of Peptide Length and Neprilysin Inhibition on Biological Responses.

The overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR selectivity compared to frog BBN sequences aiming to improve pharmacokinetic profiles. In the present study, we compared two differently truncated human endogenous GRP motifs: GRP(14–27) and GRP(18–27). An acyclic tetraamine was coupled at the N-terminus to allow for stable binding of the SPECT radionuclide 99mTc. Their biological profiles were compared in PC-3 cells and in mice without or with coinjection of phosphoramidon (PA) to induce transient neprilysin (NEP) inhibition in vivo. The two 99mTc-N4-GRP(14/18–27) radioligands displayed similar biological behavior in mice. Coinjection of PA exerted a profound effect on in vivo stability and translated into notably improved radiolabel localization in PC-3 experimental tumors. Hence, this study has shown that promising 99mTc-radiotracers for SPECT imaging may indeed derive from human GRP sequences. Radiotracer bioavailability was found to be of major significance. It could be improved during in situ NEP inhibition resulting in drastically enhanced uptake in GRPR-expressing lesions.

Peptide Controlled Shaping of Biomineralized Tin(II) Oxide into Flower-Like Particles.

The size and morphology of metal oxide particles have a large impact on the physicochemical properties of these materials, e.g., the aspect ratio of particles affects their catalytic activity. Bioinspired synthesis routes give the opportunity to control precisely the structure and aspect ratio of the metal oxide particles by bioorganic molecules, such as peptides. This study focusses on the identification of tin(II) oxide (tin monoxide, SnO) binding peptides, and their effect on the synthesis of crystalline SnO microstructures. The phage display technique was used to identify the 7-mer peptide SnBP01 (LPPWKLK), which shows a high binding affinity towards crystalline SnO. It was found that the derivatives of the SnBP01 peptide, varying in peptide length and thus in their interaction, significantly affect the aspect ratio and the size dimension of mineralized SnO particles, resulting in flower-like morphology. Furthermore, the important role of the N-terminal leucine residue in the peptide for the strong organic–inorganic interaction was revealed by FTIR investigations. This bioinspired approach shows a facile procedure for the detailed investigation of peptide-to-metal oxide interactions, as well as an easy method for the controlled synthesis of tin(II) oxide particles with different morphologies.

Study on the Materials Formed by Self-Assembling Hydrophobic, Aromatic Peptides Dedicated to Be Used for Regenerative Medicine.

The aims of this study were to identify the short aromatic peptides which are able to form highly ordered amyloid-like structures in self-assembling processes, to test the influence of length of hydrophobic peptides on tendency to aggregation, and to check if aggregated peptides fulfill requirements expected for materials useful for scaffolding. All tested hydrophobic peptides were prepared on solid phase by using DMT/NMM/TsO- as a coupling reagent. The progress of aggregation was studied by set of independent tests. All aggregated peptides were found stable under in vitro conditions. All fibrous material formed by self-assembling of peptides does not show any cytotoxic effects on L929 fibroblast cells. Peptides containing tyrosine and tryptophan residues even effectively accelerated the proliferation and stimulated the activity of L929 fibroblasts.

FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device.

Ligase-Controlled Cyclo-oligomerization of Peptides.

A biomimetic one-step ligase-catalyzed cyclo-oligomerization mediated by butelase 1, an Asn/Asp-specific ligase, is introduced that is time-, concentration-, length-, and sequence-dependent. This reaction yields cyclic mono-, di-, tri-, and tetramers from peptide precursors containing 3-15 amino acids ended with Asn and a His-Val tail. The cyclomonomers were favored when the peptide lengths were >9 amino acids. A turn-forming Pro residue at the P2 position favored the formation of higher-order cyclo-oligomers.

Benchmarking of different molecular docking methods for protein-peptide docking

BackgroundMolecular docking studies on protein-peptide interactions are a challenging and time-consuming task because peptides are generally more flexible than proteins and tend to adopt numerous conformations. There are several benchmarking studies on protein-protein, protein-ligand and nucleic acid-ligand docking interactions. However, a series of docking methods is not rigorously validated for protein-peptide complexes in the literature. Considering the importance and wide application of peptide docking, we describe benchmarking of 6 docking methods on 133 protein-peptide complexes having peptide length between 9 to 15 residues. The performance of docking methods was evaluated using CAPRI parameters like FNAT, I-RMSD, L-RMSD.ResultFirstly, we performed blind docking and evaluate the performance of the top docking pose of each method. It was observed that FRODOCK performed better than other methods with average L-RMSD of 12.46 Å. The performance of all methods improved significantly for their best docking pose and achieved highest average L-RMSD of 3.72 Å in case of FRODOCK. Similarly, we performed re-docking and evaluated the performance of the top and best docking pose of each method. We achieved the best performance in case of ZDOCK with average L-RMSD 8.60 Å and 2.88 Å for the top and best docking pose respectively. Methods were also evaluated on 40 protein-peptide complexes used in the previous benchmarking study, where peptide have length up to 5 residues. In case of best docking pose, we achieved the highest average L-RMSD of 4.45 Å and 2.09 Å for the blind docking using FRODOCK and re-docking using AutoDock Vina respectively.ConclusionThe study shows that FRODOCK performed best in case of blind docking and ZDOCK in case of re-docking. There is a need to improve the ranking of docking pose generated by different methods, as the present ranking scheme is not satisfactory. To facilitate the scientific community for calculating CAPRI parameters between native and docked complexes, we developed a web-based service named PPDbench (http://webs.iiitd.edu.in/raghava/ppdbench/).

Membrane-mediated interaction of amphipathic peptides can be described by a one-dimensional approach.

Amphipathic alpha-helical peptides, among other peripheral components of plasma membranes, are promising antimicrobial agents. Partial incorporation of a peptide into a lipid monolayer causes elastic deformations. Deformations induced by two peptides distant from each other are independent; when peptides get closer, interference between the deformations causes effective lateral interaction. We quantified the energy of membrane deformations for arbitrary configuration of two amphipathic peptides on the membrane surface. The global minimum of the deformation energy proved to be achieved when two parallel peptides are in registry at the distance of about 6 nm between the axes of peptides. The energy calculated in the unidimensional approach provides a good approximation for the dependence of the energy of peptides being in the registered configuration upon the distance between them, valid for a broad range of peptide lengths. The effective interactional length of peptides for the unidimensional approach is close to their actual length. If two parallel peptides are shifted along their axes with respect to each other, the interaction energy is also well approximated by the unidimensional potential, within the projection of one peptide onto the other. In the case when the axes of alpha helices cross at a substantial angle, the main contribution to peptide interactions comes from their edges: the effective length of peptides for the unidimensional approach is almost equal to the characteristic length of decay of deformations. Based on the results we obtained it can be concluded that interaction of membrane inclusions is quite adequately described by the potential calculated in the unidimensional approach.

A universal method for the functionalization of dyed magnetic microspheres with peptides.

The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits 'click' chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. All optimization studies were performed using a Luminex 200 assay platform, but the functionalized microspheres are capable of use in any similar multiplex format.

Upgrading collagenous smooth hound by-products: Effect of hydrolysis conditions, in vitro gastrointestinal digestion and encapsulation on bioactive properties

Collagenous by-products derived from smooth hound (Mustelus mustelus) were studied using trypsin hydrolysis to obtain protein hydrolysates with ACE (angiotensin converting enzyme) and/or PEP (prolyl endopeptidase)-inhibitory activities. Most of the hydrolysates were mainly composed of short peptides. A response surface methodology (RSM) was used to optimize the conditions of hydrolysis (pH 7, 8 or 9, and temperatures of 35, 45 or 55 °C), which led to a higher degree of hydrolysis (DH = 3.4%), as well as to an increase in ACE and PEP-inhibitory activities (maximal inhibitions of 68.1 and 81.7%, respectively). According to the model used, neither hydrolysis temperature nor pH in the ranges studied had a significant effect on DH, whereas PEP-inhibitory activity was significantly affected by both factors. pH had a significant effect on ACE-inhibitory activity. Optimum conditions of hydrolysis to obtain maximum activity differed for the two activities: 35 °C/pH 7 for PEP-inhibitory activity and 45.9 °C/pH 9 for ACE-inhibitory activity. The results suggested that these biological activities were influenced by peptide sequence rather than peptide length. This was also confirmed by the amino acid composition and molecular weight (MW) profiles. The ACE-inhibitory activity of the most potent hydrolysate was evaluated after in vitro gastrointestinal digestion. The results showed a worsening of the activity, which was related to small peptide losses and to the appearance of new low MW peptides. Hydrolysate encapsulation using an alginate-whey protein isolate microsphere improved the ACE-inhibitory activity (IC50 = 0.24 mg/ml).

Quantitation of Glycopeptides by ESI/MS - size of the peptide part strongly affects the relative proportions and allows discovery of new glycan compositions of Ceruloplasmin.

Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.

LipoFNT: Lipoylation Sites Identification with Flexible Neural Tree

Lysine lipoylation is a special type of posttranslational modification in both prokaryotes’ and eukaryotes’ proteomics researches. Such a modification takes part in several significant biological processions and plays a key role in the cellular level. In order to construct and design an accurate classification algorithm for identifying lipoylation sites in the protein level, the computational approaches should be taken into account in this field. Meanwhile, several factors plays different role in the identification of modification sites. Considering such a situation, the foundational elements of the effective identification of modification sites are the available feature description and the high effective classification. With these two elements, the distinguishing between the lipoylation samples and the nonlipoylation samples can be treated as a typical classification issue in the field of machine learning. In this work, we have proposed a method named LipoFNT, which employed the two featuring sets, including the Position‐Specific Scoring Matrix and bi‐profile Bayesian, as the classification features. And then, the flexible neural tree algorithm is utilized to deal with the imbalance classification issue in lipoylation modification sample dataset. The proposed method can achieve 81.07% in sn%, 80.29% in sp, 80.68% in Acc, 0.8076 in F1, and 0.6136 in MCC, respectively. Meanwhile, we have demonstrated the relationship between the lengths of peptide and identification of modification sites.

Short, symmetric-helical peptides have narrow-spectrum activity with low resistance potential and high selectivity.

Broad-spectrum antibiotics have, until now, been the mainstay of antibiotic therapy. However, the increasing threat of drug-resistant bacteria and the ecological imbalance of normal microbial communities have forced a reconsideration of the best strategies to treat such pathogens. Therefore, antibacterial agents with specific abilities of eliminating pathogens may provide long-term protection. Antimicrobial peptides (AMPs), which can be optimized by modifying their primary sequences, are regarded as potentially valuable in development of pathogen-specific agents. To obtain efficient narrow-spectrum AMPs, database-filtering technology, which filters the most probable amino acid composition, positive charge, sequence length and hydrophobic content of peptides against Gram-negative bacteria, was taken as the first step. Then, the filtered parameters were distributed and modified into an α-helical symmetrical structure by considering the structure-function relationship of synthesized antimicrobial peptides. Finally, short, safe and stable peptides against Escherichia coli, Salmonella pullorum and Pseudomonas aeruginosa were successfully identified. The potential peptides F1 and F4 showed low cell toxicity, low resistance potential and low salt sensitivity. CD spectroscopy of the peptides illustrated that F1 and F4 exhibited a tendency towards an α-helical structure in a membrane-mimetic environment. Indeed, fluorescence spectroscopy and electron microscopy analyses indicated that the shorter potential sequence F4 killed the bacteria by causing physical destruction of the bacterial membrane and cytosol leakage. In the mouse model test, F4 reduced the bacterial load in major organs and the cytokine (TNF-α, IL-6, and IL-1β) levels in serum significantly (P < 0.05). Collectively, this symmetric-helical distribution, dependent on database-filtering parameters, is a promising strategy for designing effective smart AMPs with high cell selectivity, and it also provides new insights into the design and optimization of pathogen-specific biomaterials.

Peptide-based materials for cancer immunotherapy.

Peptide-based materials hold great promise as immunotherapeutic agents for the treatment of many malignant cancers. Extensive studies have focused on the development of peptide-based cancer vaccines and delivery systems by mimicking the functional domains of proteins with highly specific immuno-regulatory functions or tumor cells fate controls. However, a systemic understanding of the interactions between the different peptides and immune systems remains unknown. This review describes the role of peptides in regulating the functions of the innate and adaptive immune systems and provides a comprehensive focus on the design, categories, and applications of peptide-based cancer vaccines. By elucidating the impacts of peptide length and formulations on their immunogenicity, peptide-based immunomodulating agents can be better utilized and dramatic breakthroughs may also be realized. Moreover, some critical challenges for translating peptides into large-scale synthesis, safe delivery, and efficient cancer immunotherapy are posed to improve the next-generation peptide-based immunotherapy.

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.