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Posttranslational modifications can alter protein structures, functions and locations, and are important cellular regulatory and signalling mechanisms. Spectroscopic techniques such as nuclear magnetic resonance, infrared and Raman spectroscopy, as well as small-angle scattering, can provide insights into the structural and dynamic effects of protein posttranslational modifications and their impact on interactions with binding partners. However, heterogeneity of modified proteins from natural sources and spectral complexity often hinder analyses, especially for large proteins and macromolecular assemblies. Selective labelling of proteins with stable isotopes can greatly simplify spectra, as one can focus on labelled residues or segments of interest. Employing chemical biology tools for modifying and isotopically labelling proteins with atomic precision provides access to unique protein samples for structural biology and spectroscopy. Here, we review site-specific and segmental isotope labelling methods that are employed in combination with chemical and enzymatic tools to access posttranslationally modified proteins. We discuss illustrative examples in which these methods have been used to facilitate spectroscopic studies of posttranslationally modified proteins, providing new insights into biology., Selective isotope labeling facilitates the analysis of effects of posttranslational modifications on protein structure and function.

Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so‐called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)‐adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET‐formation. Moreover, we characterized the mechanism leading to alum‐induced NET‐release in human neutrophils as rapid, NADPH oxidase‐independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+‐flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET‐release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum‐containing vaccines in humans and provides novel insights into bioenergetic requirements of NET‐formation.

Background: Peptide-based pharmaceuticals have recently experienced a renaissance due to their ability to fill the gap between the two main classes of available drugs, small molecules and biologics. Peptides combine the high potency and selectivity typical of large proteins with some of the characteristic advantages of small molecules such as synthetic accessibility, stability and the potential of oral bioavailability. Methods: In the present manuscript we review the recent literature on selected peptide-based approaches for cancer treatment, emphasizing recent advances, advantages and challenges of each strategy. Results: One of the applications in which peptide-based approaches have grown rapidly is cancer therapy, with a focus on new and established targets. We describe, with selected examples, some of the novel peptide-based methods for cancer treatment that have been developed in the last few years, ranging from naturally-occurring and modified peptides to peptidedrug conjugates, peptide nanomaterials and peptide-based vaccines. Conclusion: This review brings out the emerging role of peptide-based strategies in oncology research, critically analyzing the advantages and limitations of these approaches and the potential for their development as effective anti-cancer therapies.

Lipidation is a ubiquitous modification of peptides and proteins that can occur either co- or post-translationally. An array of different lipid classes can adorn proteins and has been shown to influence a number of crucial biological activities, including the regulation of signaling, cell-cell adhesion events, and the anchoring of proteins to lipid rafts and phospholipid membranes. Lipid modifications discovered to date include prenylation, such as S -farnesylation and S -geranylgeranylation, S - and N -palmitoylation, N -myristoylation, as well as the attachment of cholesterol, glycosylphosphatidylinositol (GPI) and phosphatidylethanolamine (PE) anchors to the C-terminus of proteins. While nature employs a range of enzymes to install lipid modifications onto proteins, the use of these for chemoenzymatic generation of lipidated proteins is often inefficient or impractical. An alternative is to harness the power of modern synthetic and semisynthetic technologies to access lipid-modified proteins in pure and homogeneously modified form. This review aims to highlight significant advances in the development of lipidation and ligation chemistry and their implementation in the synthesis and semisynthesis of homogeneously lipidated proteins that have enabled the influence of these modifications on protein structure and function to be uncovered.

Most eukaryotic proteins are modified during and/or after translation, regulating their structure, function and localisation. The role of posttranslational modifications (PTMs) in both normal cellular processes and in diseases is already well recognised and methods for detection of PTMs and generation of specifically modified proteins have developed rapidly over the last decade. However, structural consequences of PTMs and their specific effects on protein dynamics and function are not well understood. Furthermore, while random coil NMR chemical shifts of the 20 standard amino acids are available and widely used for residue assignment, dihedral angle predictions and identification of structural elements or propensity, they are not available for most posttranslationally modified amino acids. Here, we synthesised a set of random coil peptides containing common naturally occurring PTMs and determined their random coil NMR chemical shifts under standardised conditions. We highlight unique NMR signatures of posttranslationally modified residues and their effects on neighbouring residues. This comprehensive dataset complements established random coil shift datasets of the 20 standard amino acids and will facilitate identification and assignment of posttranslationally modified residues. The random coil shifts will also aid in determination of secondary structure elements and prediction of structural parameters of proteins and peptides containing PTMs. Electronic supplementary material The online version of this article (10.1007/s10858-019-00270-4) contains supplementary material, which is available to authorized users.

Immune system engagers (ISErs) make up a new class of immunotherapeutics against cancer. They comprise two or more tumor-targeting peptides and an immune-stimulating effector peptide connected by inert polymer linkers. They are produced by solid phase peptide synthesis and share the specific targeting activities of antibodies (IgGs) but are much smaller in size and exploit a different immune-stimulating mechanism. Two ISErs (Y-9 and Y-59) that bind to the cancer cell markers integrin α3 and EphA2, respectively, are analyzed here with respect to their immune cell stimulation. We have previously shown that they activate formyl peptide receptors on myeloid immune cells and induce respiratory burst in neutrophils and myeloid chemotaxis in solution. It remained, however, unclear whether these molecules can stimulate immune cells while bound to tumor cells, an essential step in the hypothesized mode of action. Here, we demonstrate that ISEr Y-9 induced respiratory burst and caused a change in the shape of neutrophils when bound to the surface of protein A beads as a model of tumor cells. More importantly, tumor cell lines carrying receptor-bound Y-9 or Y-59 also activated neutrophils, evidenced by a significant change in shape. Interestingly, similar activation was induced by the supernatants of the cells incubated with ISEr, indicating that ISErs released from tumor cells, intact or degraded into fragments, significantly contributed to immune stimulation. These findings provide new evidence for the mode of action of ISErs, namely by targeting cancer cells and subsequently provoking an innate immune response against them.

Encapsulated enzymes are stable under various conditions and used in enzyme therapy, catalysis, and biosensors. The capsules are often inspired by structures from nature such as viral capsids, DNA motifs and diatom frustules. They are based on inorganic minerals as well as soft or polymeric materials, or even a combination of these. The choice of material influences the enzyme loading and response to heat, pH and presence of proteases. This review provides a comparison of enzyme encapsulation based on these different principles with a focus on materials inspired by nature.

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.

Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. The development of various β-mercapto amino acid building blocks coupled with ligation-desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation-desulfurization sequence.

R5 is a synthetic silaffin peptide derived from diatoms that can precipitate monodisperse silica particles (SPs) from silicic acid under mild biomimetic conditions. Due to the range of potential applications, it is important to understand the mechanisms that govern such silica precipitation, however to date these mechanisms remain ambiguous. Here, we report that the N -terminal residues of R5 substantially impact the silica precipitating mechanism, most likely by affecting the self-assembling properties of the peptide. Our results show that the morphology of silaffin-derived SPs can be modulated by changing the N -terminus of the peptide.

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α3 receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

A prominent target of monoclonal antibodies as targeted therapies for cancer is the epidermal growth factor receptor, which is overexpressed on the surface of various cancer cell types. Its natural binder, the epidermal growth factor (EGF), is a 53 amino acid polypeptide. Anticancer synthetic targeted immune system engagers (ISErs) comprising two binder' peptides, which are attached to a scaffold conveying immune stimulating effector' properties, via monodisperse polyethylene glycol chains. So far, preparation of ISErs has been limited to the use of small peptides (8-20 amino acids) as binding functionalities, and they have been entirely synthesized by solid phase peptide synthesis. Here, we describe a synthetic and a semisynthetic approach for the preparation of an ISEr bearing two murine EGF molecules as binding entities (ISEr-EGF(2)). EGF was either synthesized in segments by solid phase peptide synthesis or expressed recombinantly and ligated to the scaffold by native chemical ligation. We report the successful generation of synthetic and semisynthetic ISEr-EGF(2) as well as several challenges encountered during the synthesis and ligations. We demonstrate the application of native chemical ligation for the design of larger ISEr constructs, facilitating new objectives for the coupling of small binder peptides and larger proteins to multivalent ISEr scaffolds. Copyright (c) 2017 European Peptide Society and John Wiley & Sons, Ltd.

Long, homogeneously glycosylated peptides and proteins can be assembled from multiple segments via sequential chemoselective reactions. The efficiency of the synthesis depends on the effectiveness and number of steps and on their compatibility with glycosylation methods. Here, we present how the combination of auxiliary-mediated native chemical ligation and thioester generation via hydrazinolysis from Wang-type resin enables multiple, sequential N-to-C and C-to-N ligations. The method can be applied to glycosylated peptides and peptide α-thioesters and has the potential to be further extended to sequential glycosylation, thus paving the way to the synthesis of complex homogeneous glycoproteins. We applied this methodology to the synthesis of long MUC1 variants comprising 2, 4 and 6 tandem repeats and three O-glycosylations.

Increasing the specificity of cancer therapy, and thereby decreasing damage to normal cells, requires targeting to cancer-cell specific features. The αvβ6 integrin is a receptor involved in cell adhesion and is frequently up-regulated in cancer cells compared to normal cells. We have selected a peptide ligand reported to bind specifically to the β6 integrin and have synthesized a suite of multispecific molecules to explore the potential for targeting of cancer cells. A combination of solid-phase peptide synthesis and chemoselective ligations was used to synthesize multifunctional molecules composed of integrin-targeting peptides, cytotoxic platinum(IV) prodrugs, and fluorescent or affinity probes joined with flexible linkers. The modular synthesis approach facilitates the construction of peptide–drug conjugates with various valencies and properties in a convergent manner. The binding and specificity of the multifunctional peptide conjugates were investigated using a cell line transfected with the β6 integ...

Nonenzymatic posttranslational modifications (nPTMs) of proteins are involved in age-related, metabolic and other diseases and need to be investigated at the molecular level. Here, we describe how we used organic synthesis to enable the study of the effect of argpyrimidine (Apy), an nPTM that forms at arginine residues, on one of its target proteins. We developed an efficient approach to Apy as a universal building block for Fmoc-based solid-phase peptide synthesis that allows for the construction of peptides containing this nPTM in predetermined positions. Moreover, a straightforward one-step synthesis of protecting-group-free Apy was achieved, which enabled the preparation of gram-quantities of this noncanonical amino acid that can serve as a biomarker or a feedstock in construction of Apy-containing proteins via the expanded genetic code methods.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC) into scrapie prion protein (PrPSc) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior in vitro and in vivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed., The key pathophysiologic event in prion diseases is based on a conformational change of cellular (PrPC) into scrapie prion protein (PrPSc) and is closely linked to posttranslational modifications (PTMs). Semisynthesis offers a unique opportunity to study the impact of PTMs on prion conversion, transmission, and pathogenicity, which is the major focus of this review.

Non-enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification.

The properties of elastin-like polypeptides (ELPs), specifically the fact that they are soluble in aqueous buffers below and aggregate reversibly above a well-defined transition temperature, are extensively used for protein purification, enzyme recycling, and more recently, for in vivo applications such as drug delivery and tissue engineering. ELPs are artificial but biocompatible polypeptides composed of pentameric repeats (Val-Pro-Gly-Xaa-Gly) containing different guest residues Xaa, derived from mammalian elastin. The temperature-dependent aggregation and desaggregation of ELPs is controlled by composition of the pentameric repeats as well as the number of repetitive units within the ELP. External parameters such as ELP concentration, pH, and most importantly, salt effects heavily influence the transition temperature. Here, we explore the chemical synthesis of a series of 51mer peptides consisting of 10 pentameric ELP repeats with hydrophobic as well as charged guest residues such as isoleucine, leucine, alanine, lysine, and/or glutamate all prepared by Boc-based solid phase peptide synthesis. These guest residues expand the available toolbox of synthetic ELPs and provide ELPs that can be chemically modified and tuned to specific environments. An N-terminal cysteine is added allowing disulfide-based crosslinking of ELPs and to link synthetic ELPs to a recombinantly produced protein using native chemical ligation. Transition temperatures of all synthetic ELPs and the fusion construct were determined by measuring turbidity in solution and spanned a large temperature range between 25 and 70 °C, providing synthetically accessible ELPs with transition temperatures suitable for in vitro and in vivo applications. Cycling between their soluble and aggregate state has been observed at least 6 times without significant loss of material for all synthetic ELPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Dehydroascorbate is a by-product of copper-catalysed azide-alkyne click (CuAAC) reactions and also forms advanced glycation end products (AGEs) in tissues undergoing oxidative stress. Here we isolate and characterize an arginine-dehydroascorbate adduct formed during CuAAC reactions, investigate strategies for preventing its formation, and propose its biological relevance as an AGE.

Research aimed at understanding the specific role of glycosylation patterns in protein function would greatly benefit from additional approaches allowing direct access to homogeneous glycoproteins. Here the development and application of an efficient approach for the synthesis of complex homogenously glycosylated peptides based on a multifunctional photocleavable auxiliary is described. The presence of a PEG polymer within the auxiliary enables sequential enzymatic glycosylation and straightforward isolation in excellent yields. The auxiliary-modified peptides can be directly used in native chemical ligations with peptide thioesters easily obtained via direct hydrazinolysis of the respective glycosylated peptidyl resins and subsequent oxidation. The ligated glycopeptides can be smoothly deprotected via UV irradiation. Here we apply this approach to the preparation of variants of the epithelial tumor marker MUC1 carrying one or more Tn, T or sialyl-T antigens.

Peptide hydrazides are valuable building blocks in peptide and protein chemistry, e.g. as precursors of peptide thioesters that allow the preparation of these important intermediates under mild conditions. Additional robust and versatile methods for the generation of peptide hydrazides from standard solid supports are therefore highly desired in order to facilitate access to peptide thioester via Fmoc-based SPPS. Here, the efficient generation of peptide hydrazides from conventional 4-hydroxymethyl phenol Wang-TentalGel peptidyl resins is described. Direct hydrazinolysis of a 19mer mucin1 peptide gives the protected peptide hydrazide in excellent yields. Testing a series of octapeptides carrying the 20 common proteinogenic amino acids at their C-terminus led to preparation of all corresponding peptide hydrazides in very good to excellent yields and purities. The available set of octapeptides allowed analyzing the influence of the nature of the C-terminal amino acid and of the solvent on the hydrazinolysis reaction. Furthermore, the compatibility of the method with posttranslational modifications (here glycosylation) and with potentially sensitive functional groups in amino acid side chains makes this approach a viable alternative for obtaining peptide hydrazides. It combines the advantages of a straightforward synthesis with stereochemical stability and flexibility, as it provides easy access to the peptide acid and the peptide thioester (via the hydrazide) from the same solid support.

Immobilisation of proteins on solid supports such as silica is commonly applied to improve performance of enzymes under detrimental conditions and to allow enzyme recycling. Silica biomineralisation processes occurring in nature have recently inspired approaches towards mild, biomimetic silica formation. In diatoms, complex posttranslationally modified silaffin peptides are directly involved in formation and patterning of silica cell walls. Here, chemically modified silaffin peptides are used to establish a novel strategy for silica immobilisation of target proteins. Silaffin variants carrying different modifications are covalently linked to eGFP and thioredoxin using expressed protein ligation. Covalent eGFP- and thioredoxin-silaffin conjugates are able to efficiently precipitate silica and control silica properties by choice of different silaffin modifications leading to functional encapsulation of these proteins in silica particles. Covalent protein-silaffin conjugates lead to a distinctly more efficient and homogenous encapsulation of proteins in silica, superior to random protein entrapment resulting from simple co-precipitation. Silica-immobilised proteins are confirmed to be fully active and stabilised against denaturation.

Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-μ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension.

Semisynthesis and characterization of homogeneously mono- and di-PEGylated full length PrP variants to study the impact of PEGylation (as N-glycan mimics) on protein folding and aggregation., The prion protein (PrP) is an N-glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrPSc) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrPC) into PrPSc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179–231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

The R5 peptide is derived from silaffin peptides naturally occurring in the diatom Cylindrotheca fusiformis and exhibits outstanding activity in silica precipitation. Because of its ability to cause silicification under mild conditions, several biotechnological applications based on R5-mediated biomimetic silica formation have already been reported. Yet a more detailed understanding of the R5 peptide and its intrinsic silica precipitation activity will help the rational design of R5 peptide variants as efficient agents for defined silica precipitation. The herein presented analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation. Furthermore, a tetra amino acid motif (RRIL) has to be present within the R5 sequence, but in contrast to previous reports, we demonstrate that localization of the RRIL motif shows minor impact on silica precipitation activity but rather on morphology of the resulting silica material. The amino acid sequence of silaffin peptides is a well-balanced arrangement in terms of charges, functional groups and distances. The impact of this pattern of charges and functionalities was highlighted by the disturbed morphology of silica spheres resulting from R5 variants with scrambled sequences. A detailed understanding of the highly evolved silaffin sequence(s) will contribute to unravel the intriguing process of silica biomineralization in diatoms.

Biomimetic silica formation has attracted increasing interest over the last decade for numerous biotechnological applications due to the favorable mild reaction conditions. Inspired from silica biogenesis in diatoms, peptide variants derived from native silaffins have been used for silica formation in vitro. Here a generally applicable route for covalently linking a cargo molecule to the R5 silaffin peptide via a disulfide linkage is established. The peptide CG12AB, a peptide ligand of the epidermal growth factor receptor, was chosen as model. The ability of such silaffin-cargo conjugates to encapsulate the cargo molecule during silaffin-mediated silica precipitation is demonstrated. Cargo release from silica material under different conditions was analyzed. The results obtained here provide a rational basis for developing engineered R5 silaffin peptides into efficient tools for silica precipitation as well as for entrapment and release of cargo molecules under physiological conditions.

Hsp70s are molecular chaperones involved in the folding and assembly of proteins. They recognize hydrophobic amino acid stretches in their substrate binding groove. However, a detailed understanding of substrate specificity is still missing. Here, we use the endoplasmic reticulum-resident Hsp70 BiP to identify binding sites in a natural client protein. Two sites are mutually recognized and form stable Hsp70-substrate complexes. In silico and in vitro analyses revealed an extended substrate conformation as a crucial factor for interaction and show an unexpected plasticity of the substrate binding groove. The basic binding mechanism is conserved among different Hsp70s.

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site.

Access to integral membrane proteins via chemical synthesis or semisynthesis can provide detailed insights into the assembly and function of this diverse class of proteins. About one-quarter to one-third of all eukaryotic proteins are membrane proteins, and they fulfill a multitude of different functions as receptors and transducers for extracellular signals, as ion channels, and as scaffolds for large multi-component assemblies. Many of these are still enigmatic and could benefit from synthetic access to these proteins, which allows almost unlimited variations of the peptide chain in order to address highly specific questions regarding their folding and function. The technical and synthetic challenges encountered during synthesis of these macromolecules are tremendous and help to drive the field of chemical protein synthesis beyond current limitations with respect to the ability to synthesize “difficult” hydrophobic sequences, purify them, and handle them in subsequent transformations, such as chemoselective ligation reactions. Here we provide an account of the synthetic challenges that we encountered en route to the first total chemical synthesis of an integral membrane protein comprising three membrane-spanning domains: the E. coli diacylglycerol kinase. We hope this detailed description of our efforts will be helpful to others in tackling challenging protein synthesis targets.

Eukaryotic cell surface proteins are often modified by a glycosylphosphatidylinositol (GPI) anchor. More than 200 of these post-translationally altered proteins are presently known, a prominent example being the prion protein (PrP). Although the significance of the GPI anchor is well recognized, efforts to study its function are hampered due to its complex chemical nature, which combines hydrophilic glycosyl chains with hydrophobic lipid moieties. Here we describe a general method for the synthesis of a GPI-anchored peptide containing an N-terminal Cys. This module can be employed for the production of proteins containing a natural GPI anchor using expressed protein ligation.

Protein semisynthesis based on native chemical ligation has become a major protein engineering tool that allows manipulation of domains of proteins of all sizes. It helps to overcome limitations in chemical protein synthesis set by the inherent size limits of solid phase peptide synthesis. Here we present a semisynthesis approach that provides access to N-terminally-modified variants of human thymidine monophosphate kinase (TMPK). This enzyme is intimately involved in activating nucleoside-based drugs directed against viral infections such as HIV and against certain types of cancers. The option to chemically synthesize and manipulate the first 30 amino acids of this enzyme via protein semisynthesis allows direct substitution of vital amino acids in the P-loop of this enzyme for probing the mechanism of phosphate transfer and direct observation of substrate or inhibitor binding. Efficient native chemical ligation of two N-terminal segments, one comprising the wild type sequence and one containing a small fluorescent probe, provides milligram amounts of two semisynthetic TMPK variants. An efficient folding procedure in the presence of substrate nucleotides provides access to active semisynthetic TMPK variants.

A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into a pathogenic conformation, designated scrapie prion protein (PrP(Sc)), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrP(C) into a detergent-insoluble conformation distinct from PrP(Sc). The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie-infected cells EGCG treatment was beneficial; formation of PrP(Sc) ceased. However, in uninfected cells EGCG interfered with the stress-protective activity of PrP(C). As a consequence, EGCG-treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrP(C) to protect cells from stress and indicate efficient intracellular pathways to degrade non-native conformations of PrP(C).

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.

Folding, assembly and stability of α-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b 559 ′ was studied by fluorescence resonance energy transfer. Cytochrome b 559 ′ consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b 559 ′ monomer, each carrying a specific fluorescent dye, allowed monitoring helix–helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Our ability to alter and control the structure and function of biomolecules, and of proteins in particular, will be of utmost importance in order to understand their respective biological roles in complex systems such as living organisms. This challenge has prompted the development of powerful modern techniques in the fields of molecular biology, physical biochemistry and chemical biology. These fields complement each other and their successful combination has provided unique insights into protein structure and function at the level of isolated molecules, cells and organisms. Chemistry is without doubt most suited for introducing subtle changes into biomolecules down to the atomic level, but often struggles when it comes to large targets, such as proteins. In this review, we attempt to give an overview of modern and broadly applicable techniques that permit chemical synthesis to be applied to complex protein targets in order to gain control over their structure and function. As will be demonstrated, these approaches offer unique possibilities in our efforts to understand the molecular basis of protein functioning in vitro and in vivo. We will discuss modern synthetic reactions that can be applied to proteins and give examples of recent highlights. Another focus of this review will be the application of inteins as versatile protein engineering tools.

The elucidation of structure and function of proteins and membrane proteins by EPR spectroscopy has become increasingly important in recent years as technological advances have been made in the design of spectrometers and in the chemistry of the nitroxide group. These new developments have increased the demand for tailor-made amino acids carrying a spin label on the one hand and for reliable methods for their incorporation into proteins on the other. Here we describe methods for site-specific spin labelling of proteins. It is shown that a combination of recombinant synthesis of proteins with chemically produced peptides (expressed protein ligation) allows the preparation of site-specifically spin-labelled proteins. Copyright © 2005 John Wiley & Sons, Ltd.

Vpu, an 81-residue membrane protein encoded by the genome of HIV-1, is involved in CD4 degradation and facilitates virion budding from infected cells. The latter activity requires an intact transmembrane (TM) domain; however, the mechanism remains unclear. Vpu forms ion channels, an activity linked to the TM domain and envisioned to arise by oligomerization. The precise number of Vpu monomers that structure the channel is not yet known. To address this issue, we have synthesized tetrameric and pentameric proteins consisting of a carrier template to which four or five peptides corresponding to the TM domain of Vpu are attached. Ketoxime-forming chemoselective ligation efficiently ligated four and five copies, respectively, of the linear transmembrane peptide that was solubilized by the addition of a cleavable polyethylene glycol-polyamide auxiliary to a template. Purified tetrameric and pentameric proteins, denoted as T(4)Vpu and T(5)Vpu, exhibit the predicted mass as determined by MS analysis and fold with a high helical content as evidenced by CD. Both T(4)Vpu and T(5)Vpu, after reconstitution in lipid bilayers, form discrete ion channels of distinct conductance and high propensity to be open. The most frequent openings have a single channel conductance of 42 +/- 5 pS for T(4)Vpu and 76 +/- 5 pS for T(5)Vpu in 0.5m KCl. These findings validate the notion that the channels formed by Vpu result from the self-assembly of monomers. We conclude that a five-helix bundle of the TM of Vpu may approximate the structural motif underlying the oligomeric state of the conductive channel.

We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

Ara-adenosine (adenine 9-β-D-arabinofuranoside) and ara-guanosine (guanine 9-β-D-arabinofuranoside) are converted into 2′ halogenated ATP and GTP analogues by triflation and subsequent inversion of configuration at C-2′. For the commercially unavailable ara-guanosine a short synthesis starting from guanosine is presented. The nucleotide analogues could serve for the preparation of heavy atom derivatives of ATP- and GTP-binding proteins useful for protein crystal structure determination by MIR/MAD phasing.

Many cellular processes are regulated by posttranslational modifications that are recognized by specific domains in protein binding partners. These interactions are often weak, thus allowing a highly dynamic and combinatorial regulatory network of protein-protein interactions. We report an efficient strategy that overcomes challenges in structural analysis of such a weak transient interaction between the Tudor domain of the Survival of Motor Neuron (SMN) protein and symmetrically dimethylated arginine (sDMA). The posttranslational modification is chemically introduced and covalently linked to the effector module by a one-pot expressed protein ligation (EPL) procedure also enabling segmental incorporation of NMR-active isotopes for structural analysis. Covalent coupling of the two interacting moieties shifts the equilibrium to the bound state, and stoichiometric interactions are formed even for low affinity interactions. Our approach should enable the structural analysis of weak interactions by NMR or X-ray crystallography to better understand the role of posttranslational modifications in dynamic biological processes.