Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.     

Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds

Cyclic disulfide‐rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X‐ray structures of these peptides to assist in drug design applications, but disulfide‐rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L ‐ and D ‐forms of three prototypic cyclic disulfide‐rich peptides: SFTI‐1 (14‐mer with one disulfide bond), cVc1.1 (22‐mer with two disulfide bonds), and kB1 (29‐mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi‐racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.     

Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABAB Receptor Activation Reveal a Minimal Functional Motif

α‐Conotoxins are disulfide‐rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α‐conotoxins that also modulate voltage‐gated calcium channels by acting as G protein‐coupled GABA_B receptor (GABA_BR) agonists. These α‐conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α‐conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α‐conotoxins known to inhibit high voltage‐activated calcium channels via GABA_BR activation. Remarkably, all disulfide isomers of the active α‐conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.     

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine‐rich snakin‐1 and ‐2 antimicrobial peptides by using a combination of solid‐phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40–50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin‐2 compared to natural snakin‐2, we demonstrated that synthetic snakin‐2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin‐2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.     

Basic Amino Acid Conjugates of 1,2‐Diselenan‐4‐amine with Protein Disulfide Isomerase‐like Functions as a Manipulator of Protein Quality Control

Protein disulfide isomerase (PDI) can assist immature proteins to correctly fold by controlling cysteinyl disulfide (SS)‐relating reactions (i. e., SS‐formation, SS‐cleavage, and SS‐isomerization). PDI controls protein quality by suppressing protein aggregation, as well as functions as an oxidative folding catalyst. Following the amino acid sequence of the active center in PDI, basic amino acid conjugates of 1,2‐diselenan‐4‐amine ( 1 ), which show oxidoreductase‐ and isomerase‐like activities for SS‐relating reactions, were designed as a novel PDI model compound. By conjugating the amino acids, the diselenide reduction potential of compound 1 was significantly increased, causing improvement of the catalytic activities for all SS‐relating reactions. Furthermore, these compounds, especially histidine‐conjugated one, remarkably suppressed protein aggregation even at low concertation (0.3 mM～). Thus, it was demonstrated that the conjugation of basic amino acids into 1 simultaneously achieves the enhancement of the redox reactivity and the capability to suppress protein aggregation.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Intracellular Delivery of Native Proteins Facilitated by Cell‐Penetrating Poly(disulfide)s

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis‐independent delivery of proteins to live mammalian cells are reported. By using either a “glycan” tag naturally derived from glycosylated proteins or a “traceless” tag that could reversibly label native lysines on non‐glycosylated proteins, followed by bioorthogonal conjugation with cell‐penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their “native” functional forms with immediate bioavailability.     

Chemical cross‐linking with a diazirine photoactivatable cross‐linker investigated by MALDI‐ and ESI‐MS/MS

Crystallography and nuclear magnetic resonance are well‐established methods to study protein tertiary structure and interactions. Despite their usefulness, such methods are not applicable to many protein systems. Chemical cross‐linking of proteins coupled with mass spectrometry allows low‐resolution characterization of proteins and protein complexes based on measuring distance constraints from cross‐links. In this work, we have investigated cross‐linking by means of a heterobifunctional cross‐linker containing a traditional N‐hydroxysuccinimide (NHS) ester and a UV photoactivatable diazirine group. Activation of the diazirine group yields a highly reactive carbene species, with potential to increase the number of cross‐links compared with homobifunctional, NHS‐based cross‐linkers. Cross‐linking reactions were performed on model systems such as synthetic peptides and equine myoglobin. After reduction of the disulfide bond, the formation of intra‐ and intermolecular cross‐links was identified and the peptides modified with both NHS and diazirine moieties characterized. Fragmentation of these modified peptides reveals the presence of a marker ion for intramolecular cross‐links, which facilitates identification. Copyright © 2010 John Wiley & Sons, Ltd.     

High‐throughput method for on‐target performic acid oxidation of MALDI‐deposited samples

An information‐rich on‐target performic acid oxidation method, which is compatible with alkylation for differentiation of free cysteine versus disulfide‐containing peptides, is described. On‐target oxidation is achieved using performic acid vapor to oxidize disulfide‐containing peptides and/or small proteins on the matrix‐assisted laser desorption/ionization (MALDI) sample deposits. The on‐target oxidation method is preferred over solution‐phase oxidation methods because (1) less sample handing is required, (2) oxidation throughput is drastically increased and (3) ion suppression effects are reduced because performic acid is not added directly to the MALDI spot. The utility of this method is demonstrated by simultaneous oxidation of multiple MALDI sample deposits containing model disulfide‐linked peptides, intact bovine insulin and a bovine ribonuclease A proteolytic digest. Copyright © 2009 John Wiley & Sons, Ltd.     

Functional and Photochemical Characterization of a Light‐Driven Proton Pump from the Gammaproteobacterium Pantoea vagans

Photoactive retinal proteins are widely distributed throughout the domains of the microbial world (i.e., bacteria, archaea, and eukarya). Here we describe three retinal proteins belonging to a phylogenetic clade with a unique DTG motif. Light‐induced decrease in the environmental pH and its inhibition by carbonyl cyanide m‐chlorophenylhydrazone revealed that these retinal proteins function as light‐driven outward electrogenic proton pumps. We further characterized one of these proteins, Pantoea vagans rhodopsin (PvR), spectroscopically. Visible spectroscopy and high‐performance liquid chromatography revealed that PvR has an absorption maximum at 538 nm with the retinal chromophore predominantly in the all‐trans form (>90%) under both dark and light conditions. We estimated the pK_a values of the protonated Schiff base of the retinal chromophore and its counterion as approximately 13.5 and 2.1, respectively, by using pH titration experiments, and the photochemical reaction cycle of PvR was measured by time‐resolved flash‐photolysis in the millisecond timeframe. We observed a blue‐shifted and a red‐shifted intermediate, which we assigned as M‐like and O‐like intermediates, respectively. Decay of the M‐like intermediate was highly sensitive to environmental pH, suggesting that proton uptake is coupled to decay of the M‐like intermediate. From these results, we propose a putative model for the photoreaction of PvR.     

Silk‐Inspired β‐Peptide Materials Resist Fouling and the Foreign‐Body Response

The functions of implants like medical devices are often compromised by the host's foreign‐body response (FBR). Herein, we report the development of low‐FBR materials inspired by serine‐rich sericin from silk. Poly‐β‐homoserine (β‐HS) materials consist of the hydrophilic unnatural amino acid β‐homoserine. Self‐assembled monolayers (SAMs) of β‐HS resist adsorption by diverse proteins, as well as adhesion by cells, platelets, and diverse microbes. Experiments lasting up to 3 months revealed that, while implantation with control PEG hydrogels induced obvious inflammatory responses, collagen encapsulation, and macrophage accumulation, these responses were minimal with β‐HS hydrogels. Strikingly, the β‐HS hydrogels induce angiogenesis in implant‐adjacent tissues. Molecular dynamics simulations indicated that the low FBR performance of β‐HS results from what we term “dual hydrogen bonding hydration”, wherein both the backbone amide groups and the sidechain hydroxyl groups of β‐HS undergo hydration.     

Stabilization of the Cysteine‐Rich Conotoxin MrIA by Using a 1,2,3‐Triazole as a Disulfide Bond Mimetic

The design of disulfide bond mimetics is an important strategy for optimising cysteine‐rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4‐disubstituted‐1,2,3‐triazole bridge, are described. Sequential copper‐catalyzed azide–alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole–disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4–Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.     

Nanoengineering of Stimuli‐Responsive Protein‐Based Biomimetic Protocells as Versatile Drug Delivery Tools

We present a general strategy to nanoengineer protein‐based colloidal spheres (biomimetic protocells) as versatile delivery carriers with stimuli responsiveness by the electrostatic assembly of binary components (proteins and polypeptides) in association with intermolecular disulfide cross‐linking. The size of the colloidal spheres, ranging from nanoscale to microscale, is readily tuned through parameters like protein and polypeptide concentration, the ratio between both, pH, and so on. Moreover, such colloidal spheres show versatile encapsulation of various guest molecules including small organic molecules and biomacromolecules. The pH and redox dual‐responsiveness facilitates the rapid release of the payload in an acidic and reductant‐enriched ambient such as in lysosomes. Thus, nanoengineering of protein‐based biomimetic protocells opens a new alternative avenue for developing delivery vehicles with multifunctional properties towards a range of therapeutic and diagnostic applications.     

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic‐binding proteins is important for understanding arsenic health effects and for developing arsenic‐based therapeutics. We report here a strategy for the capture and identification of arsenic‐binding proteins in living cells. We designed an azide‐labeled arsenical, p‐azidophenylarsenoxide (PAzPAO), to serve bio‐orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic‐binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic‐binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde‐3‐phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic‐induced health effects and in cancer treatment.     

Protein‐Structure‐Directed Metal–Organic Zeolite‐like Networks as Biomacromolecule Carriers

Fabrication of zeolite‐like metal–organic frameworks (ZMOFs) for advanced applications, such as enzyme immobilization, is of great interest but is a great synthetic challenge. Herein, we have developed a new strategy using proteins as structure‐directed agents to direct the formation of new ZMOFs that can act as versatile platforms for the in situ encapsulation of proteins under ambient conditions. Notably, protein incorporation directs the formation of a ZMOF with a sodalite ( sod ) topology instead of a non‐porous diamondoid ( dia ) topology under analogous synthetic conditions. Histidines in proteins play a crucial role in the observed templating effect. Modulating histidine content thereby influenced the resultant MOF product (from dia to dia + sod mixture and, ultimately, to sod MOF). Moreover, the resulting ZMOF‐incorporated proteins preserved their activity even after exposure to high temperatures and organic solvents, demonstrating their potential for biocatalysis and biopharmaceutical applications.     

Study of the Function of G‐Rich Aptamers Selected for Lung Adenocarcinoma

Guanine (G)‐rich oligonucleotides have attracted considerable interest as therapeutic agents. Two G‐rich aptamers were selected against epidermal growth factor receptor (EGFR)‐transfected A549 cells, and their G‐rich domains (S13 and S50) were identified to account for the binding of parental aptamers. Circular dichroism (CD) spectra showed that S13 and S50 bound to their targets by forming parallel quadruplexes. Their binding, internalization, and antiproliferation activity in cancer and noncancer cells were investigated by flow cytometry and 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) assay, and compared with those of nucleolin‐binding AS1411 and thrombin‐binding aptamer. The two truncated aptamers (S13 and S50) have good binding and internalization in cancer cells and noncancer cells; however, only S50, similar to AS1411, shows potent antiproliferation against cancer cells. Our data suggest that tumor‐selective antiproliferation of G‐rich oligonucleotides does not directly depend on the binding of the G‐rich aptamer to cells.     

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.     

Structure‐based redesign of proteins for minimal T‐cell epitope content

The protein universe displays a wealth of therapeutically relevant activities, but T‐cell driven immune responses to non‐“self” biological agents present a major impediment to harnessing the full diversity of these molecular functions. Mutagenic T‐cell epitope deletion seeks to mitigate the immune response, but can typically address only a small number of epitopes. Here, we pursue a “bottom‐up” approach that redesigns an entire protein to remain native‐like but contain few if any immunogenic epitopes. We do so by extending the Rosetta flexible‐backbone protein design software with an epitope scoring mechanism and appropriate constraints. The method is benchmarked with a diverse panel of proteins and applied to three targets of therapeutic interest. We show that the deimmunized designs indeed have minimal predicted epitope content and are native‐like in terms of various quality measures, and moreover that they display levels of native sequence recovery comparable to those of non‐deimmunized designs. © 2013 Wiley Periodicals, Inc.     

Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

During SDS‐PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ～110 residues‐long, sequence‐homologous, structurally‐analogous, extracellular domains of human E‐ and N‐cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively‐charged and positively‐charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater‐than‐expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS‐protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.