Mechanical properties that influence antimicrobial peptide activity in lipid membranes

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.     

Composition of the peptide fraction in human blood plasma: database of circulating human peptides

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20 000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15 000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, β2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aα-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.     

On the design and efficacy assessment of self‐assembling peptide‐based hydrogel‐glycosaminoglycan mixtures for potential repair of early stage cartilage degeneration

Peptide‐based hydrogels are of interest for their potential use in regenerative medicine. Combining these hydrogels with materials that may enhance their physical and biological properties, such as glycosaminoglycans, has the potential to extend their range of biomedical applications, for example in the repair of early cartilage degeneration. The aim of this study was to combine three self‐assembling peptides (P₁₁‐4, P₁₁‐8, and P₁₁‐12) with chondroitin sulphate at two molar ratios of 1:16 and 1:64 in 130 and 230 mM Na⁺ salt concentrations. The study investigates the effects of mixing self‐assembling peptide and glycosaminoglycan on the physical and mechanical properties at 37°C. Peptide alone, chondroitin sulphate alone, and peptide in combination with chondroitin sulphate were analysed using Fourier transform infrared spectroscopy to determine the β‐sheet percentage, transmission electron microscopy to determine the fibril morphology, and rheology to determine the elastic and viscous modulus of the materials. All of the variables (peptide, salt concentration, and chondroitin sulphate molar ratio) had an effect on the mechanical properties, β‐sheet formation, and fibril morphology of the hydrogels. P₁₁‐4 and P₁₁‐8‐chondroitin sulphate mixtures, at both molar ratios, were shown to have a high β‐sheet percentage, dense entangled fibrillar networks, as well as high mechanical stiffness in both (130 and 230 mM) Na⁺ salt solutions when compared with the P₁₁‐12/chondroitin sulphate mixtures. These peptide/chondroitin sulphate hydrogels show promise for biomedical applications in glycosaminoglycan depleted tissues.     

Viscoelastic properties and nanoscale structures of composite oligopeptide-polysaccharide hydrogels

Biocompatible and biodegradable peptide hydrogels are drawing increasing attention as prospective materials for human soft tissue repair and replacement. To improve the rather unfavorable mechanical properties of our pure peptide hydrogels, in this work we examined the possibility of creating a double hydrogel network. This network was created by means of the coassembly of mutually attractive, but self-repulsive oligopeptides within an already-existing fibrous network formed by the charged, biocompatible polysaccharides chitosan, alginate, and chondroitin. Using dynamic oscillatory rheology experiments, it was found that the coassembly of the peptides within the existing polysaccharide network resulted in a less stiff material as compared to the pure peptide networks (the elastic modulus G' decreased from 90 to 10 kPa). However, these composite oligopeptide-polysaccharide hydrogels were characterized by a greater resistance to deformation (the yield strain γ grew from 4 to 100%). Small-angle neutron scattering (SANS) was used to study the 2D cross-sectional shapes of the fibers, their dimensional characteristics, and the mesh sizes of the fibrous networks. Differences in material structures found with SANS experiments confirmed rheology data, showing that incorporation of the peptides dramatically changed the morphology of the polysaccharide network. The resulting fibers were structurally very similar to those forming the pure peptide networks, but formed less stiff gels because of their markedly greater mesh sizes. Together, these findings suggest an approach for the development of highly deformation-resistant biomaterials.     

Morphology of artificial silica matrices formed via autosilification of a silaffin/protein polymer chimera

Protein polymers (long-chain proteins in which a specific amino acid sequence "monomer" is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK 1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK 1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.     

Bioinspired polymers that control intracellular drug delivery

One of the important characteristics of biological systems is their ability to change important properties in response to small environmental signals. The molecular mechanisms that biological molecules utilize to sense and respond provide interesting models for the development of “smart” polymeric biomaterials with biomimetic properties. An important example of this is the protein coat of viruses, which contains peptide units that facilitate the trafficking of the virus into the cell via endocytosis, then out of the endosome into the cytoplasm, and from there into the nucleus. We have designed a family of synthetic polymers whose compositions have been designed to mimic specific peptides on viral coats that facilitate endosomal escape. Our biomimetic polymers are responsive to the lowered pH within endosomes, leading to disruption of the endosomal membrane and release of important biomolecular drugs such as DNA, RNA, peptides and proteins to the cytoplasm before they are trafficked to lysosomes and degraded by lysosomal enzymes. In this article, we review our work on the design, synthesis and action of such smart, pH-sensitive polymers.     

Recombinant production of self-assembling SZ-structured peptides using SUMO as a fusion partner

ABSTRACT: BACKGROUND: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P11-family of peptides includes, P11-4 (QQRFEWEFEQQ) and the complementary peptides P11-13 (EQEFEWEFEQE) and P11-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P11-4) or when mixed (P11-13 and P11-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage. RESULTS: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy. CONCLUSIONS: The fusion proteins were produced in high yields and the beta-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.     

Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Optimization of hydrophobic domains in peptides that undergo transformation from alpha-helix to beta-fibril

Recent studies on peptide fibrillogenesis by the de novo method as well as amyloidogenic proteins including prion proteins and Alzheimer's beta-peptides have provided insights into the conformational changes, such as alpha-helix to beta-structure, involved in folding and misfolding processes. We have found that an exposed hydrophobic nucleation domain at N-terminal causes a structural transition of a peptide from alpha-helix to beta-fibril. It became clear that N-terminal acyl groups of particular lengths in a 2alpha-helix peptide caused the peptide to undergo an alpha-to-beta transition. The peptide with the octanoyl group (C8-2alpha) showed the highest rate of transformation. The study of the designed peptides revealed that these alpha-to-beta transitions were closely related to the initial alpha-helix conformation and its stability. Engineering peptides that undergo alpha-to-beta transitions are attractive not only to the study of pathogenic proteins such as prion proteins, but also to the control of self-assembly of peptides, which will lead to the development of peptidyl self-assembling materials.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

秀丽小杆线虫分泌蛋白组的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalP v3.0、TargetP v1.01、Big-PI Predictor和TMHMM v2.0,预测了秀丽小杆线虫（Caenorthaditis elegans ws123）的全基因组19855个ORF编码蛋白的信号肽,同时系统分析了信号肽的特征。结果表明,在19855个秀丽小杆线虫的蛋白中,有1990条为带有信号肽的分泌型蛋白,其中,1936条为典型的分泌型信号肽（即信号肽酶Ⅰ型信号肽）,53条为信号肽酶Ⅱ型信号肽,1条为信号肽酶Ⅳ型信号肽;在Ⅰ型信号肽中,有41条为RR-motif亚组型信号肽。在1990条信号肽中,有742条没有典型的N-区,其余1248条包含典型的3个区。比较了秀丽小杆线虫与原核生物分泌蛋白信号肽中20种氨基酸残基在信号肽酶切位点的使用情况,表明：在Ⅰ型信号肽酶切位点,其信号肽中使用的氨基酸总体趋势与原核生物基本相似,但秀丽小杆线虫选用的氨基酸种类更多,变化更大;在Ⅱ型信号肽酶切位点,秀丽小杆线虫脂蛋白信号肽中使用的氨基酸的种类与原核生物有很大的不同。通过与真核单细胞生物比较,作为真核多细胞生物的秀丽小杆线虫,其分泌蛋白信号肽所占比例更高、种类更多,可知线虫信号肽的组成具有很高的多态性,表明该物种的分泌蛋白具有多种功能。此外,分析结果显示,脂蛋白信号肽在结构上比分泌型信号肽更为保守。在秀丽小杆线虫分泌蛋白中出现了少数氨基酸组成完全一致的信号肽,采用BLAST 2 SEQUENECES对具有相同信号肽的分泌蛋白进行了序列比对,结果表明具有相同信号肽的分泌蛋白同源性非常高,它们的存在是生物进化过程中基因倍加（duplication）及环境选择的结果,信号肽特征的详细描述必将对这些蛋白功能的研究提供重要的帮助。      

The penetrating properties of the tumor homing peptide LyP‐1 in model lipid membranes

Cell‐penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP‐1 is a cyclic 9‐amino‐acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP‐1 peptide is the linear truncated form of LyP‐1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP‐1 peptide and linear truncated tLyP‐1 peptide with model plasma membrane in order to understand their passive, energy‐independent mechanism of uptake. The experiments reveal that internalization of tLyP‐1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone‐shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP‐1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP‐1 peptide has enhanced penetrating properties compared with the cyclic LyP‐1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP‐1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.     

Cellular cross-linking of peptide modified hydrogels

Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.     

Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Control of protein functional dynamics by peptide linkers

Control of structural flexibility is essential for the proper functioning of a large number of proteins and multiprotein complexes. At the residue level, such flexibility occurs due to local relaxation of peptide bond angles whose cumulative effect may result in large changes in the secondary, tertiary or quaternary structures of protein molecules. Such flexibility, and its absence, most often depends on the nature of interdomain linkages formed by oligopeptides. Both flexible and relatively rigid peptide linkers are found in many multidomain proteins. Linkers are thought to control favorable and unfavorable interactions between adjacent domains by means of variable softness furnished by their primary sequence. Large-scale structural heterogeneity of multidomain proteins and their complexes, facilitated by soft peptide linkers, is now seen as the norm rather than the exception. Biophysical discoveries as well as computational algorithms and databases have reshaped our understanding of the often spectacular biomolecular dynamics enabled by soft linkers. Absence of such motion, as in so-called molecular rulers, also has desirable functional effects in protein architecture. We review here the historic discovery and current understanding of the nature of domains and their linkers from a structural, computational, and biophysical point of view. A number of emerging applications, based on the current understanding of the structural properties of peptides, are presented in the context of domain fusion of synthetic multifunctional chimeric proteins.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

植物多肽抗生素研究进展

植物多肽抗生素是一类对细菌、真菌等微生物及某些昆虫和动植物细胞具有抑制或杀灭作用的小分子多肽. 根据多肽抗生素的氨基酸序列及二级结构,可将植物多肽抗生素分为9类,包括硫素（thionins）、植物防御素（plant defensins）、转脂蛋白（lipid transfer proteins, LTPs）、橡胶素（heveins）、打结素（knottins）、凤仙花素(1b-AMPs)和新近发现的荠菜素（shepherdins）、蜕皮素（snakins）、环肽（cyclotides）. 对近年来植物多肽抗生素的分类、抗菌机理、生物活性及基因工程等方面的研究情况作一介绍,希望有助于我国在这一领域的研究与开发.     

A designed glycopeptide array for characterization of sugar-binding proteins toward a glycopeptide chip technology

For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as “protein fingerprints” (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies.     

Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent-resistant rafts

Specific proteins and lipids sequester to regions of cell membranes called rafts. Due to their high content of sphingomyelin (SM) and cholesterol, raft bilayers are thicker than nonraft bilayers and, at least at 4 degrees C, are resistant to Triton X-100 extraction. It has been postulated that rafts concentrate proteins with long transbilayer domains because of "hydrophobic matching" between the transbilayer domain and the thick bilayer hydrocarbon region. However, because the area compressibility and bending moduli of SM:cholesterol bilayers are larger than that of nonraft bilayers, there should be an energy cost to partition proteins or peptides into rafts. To determine the effects on peptide sorting of raft thickness and mechanical properties, we incorporated two transbilayer peptides (P-23, P-29) into bilayers composed of SM, dioleoylphosphatidylcholine, and cholesterol, separated detergent-soluble membranes (DSMs) from detergent-resistant membranes (DRMs), and measured their peptide and lipid compositions. P-23 and P-29 were designed to have transbilayer domains that matched the hydrocarbon thicknesses of DSMs and DRMs, respectively. At both 4 degrees C and 37 degrees C DSMs were enriched in dioleoylphosphatidylcholine and DRMs were enriched in SM and cholesterol. At both temperatures both P-23 and P-29 preferentially localized to DSMs, demonstrating the importance of bilayer mechanical properties relative to hydrophobic mismatch. However, at 37 degrees C significantly more P-29 than P-23 was located in DRMs, implying that hydrophobic matching played a role in peptide sorting at physiological temperature. These experiments demonstrate that the sorting of peptides as measured by detergent extraction is temperature-dependent and both bilayer mechanical properties and hydrophobic matching impact peptide distribution between DSMs and DRMs.     

Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation.

Most molecules that are not actively imported by living cells are impermeable to cell membranes, including practically all macromolecules and even many small molecules whose physicochemical properties prevent passive membrane diffusion. The use of peptide vectors capable of transporting such molecules into cells in the form of covalent conjugates has become an increasingly attractive solution to this problem. Not only has this technology permitted the study of modulating intracellular target proteins, but it has also gained importance as an alternative to conventional cellular transfection with oligonucleotides. Peptide vectors derived from viral, bacterial, insect, and mammalian proteins endowed with membrane translocation properties have now been proposed as delivery vectors. These are discussed comprehensively and critically in terms of relative utility, applications to compound classes and specific molecules, and relevant conjugation chemistry. Although in most cases the mechanisms of membrane translocation are still unclear, physicochemical studies have been carried out with a number of peptide delivery vectors. Unifying and distinguishing mechanistic features of the various vectors are discussed. Until a few years ago speculations that it might be possible to deliver peptides, proteins, oligonucleotides, and impermeable small molecules with the aid of cellular delivery peptides not only to target cells in vitro, but in vivo, was received with scepticism. However, the first studies showing pharmacological applications of conjugates between macromolecules and peptide delivery vectors are now being reported, and therapies based on such conjugates are beginning to appear feasible.