Folding Yield Research Articles

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

Optimal face-to-face coupling for fast self-folding kirigami.

Kirigami-inspired designs can enable self-folding three-dimensional materials from flat, two-dimensional sheets. Hierarchical designs of connected levels increase the diversity of possible target structures, yet they can lead to longer folding times in the presence of fluctuations. Here, we study the effect of rotational coupling between levels on the self-folding of two-level kirigami designs driven by thermal noise in a fluid. Naturally present due to hydrodynamic resistance, we find that this coupling parameter can significantly impact a structure's self-folding pathway, thus enabling us to assess the quality of a kirigami design and the possibility for its optimization in terms of its folding rate and yield.

Protein engineering of antibody fragments for pharmaceutical production

Antibody fragments without the Fc region are attracting attention in the pharmaceutical industry due to their high ability to penetrate solid tissues, cost-effective expression using microbial expression systems, and distinctive modes of action compared to those of full-size antibodies. Based on these characteristics, several antibody fragment agents have been approved. However, developing platform engineering methodologies to accelerate their development is important. In this review, we summarize and discuss protein engineering strategies for preparing therapeutic antibody fragments composed of antibody variable domains. Three (introduction of high-solubility tag systems, complementarity-determining region grafting, and domain arrangements) and two (introduction of purification tag systems and mutagenesis studies for protein L- or protein A-binding) protein engineering strategies have been reported for the cultivation and purification processes, respectively. Fusion tags might negatively impact molecular folding, function, immunogenicity, and final yield. If the production behavior of antibody fragments is not improved through complementarity-determining region grafting, domain arrangements, or human sequence-based mutagenesis, using additional fusion tag systems should be considered, with careful attention to the points described above. This summarized knowledge regarding protein engineering strategies for effectively producing antibody fragments will further accelerate therapeutic antibody fragment development.

Solid Phase Synthesis of DNA Nanostructures in Heavy Liquid.

Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules.

Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides.

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.

Precision Modification of Native Antibodies.

Antibodies, particularly of the immunoglobulin G (IgG) isotype, are a group of biomolecules that are extensively used as affinity reagents for many applications in research, disease diagnostics, and therapy. Most of these applications require antibodies to be modified with specific functional moieties, including fluorophores, drugs, and proteins. Thus, a variety of methodologies have been developed for the covalent labeling of antibodies. The most common methods stably attach functional molecules to lysine or cysteine residues, which unavoidably results in heterogeneous products that cannot be further purified. In an effort to prepare homogeneous antibody conjugates, bioorthogonal handles have been site-specifically introduced via enzymatic treatment, genetic code expansion, or genetically encoded tagging, followed by functionalization using bioorthogonal conjugation reactions. The resulting homogeneous products have proven superior to their heterogeneous counterparts for both in vitro and in vivo usage. Nevertheless, additional chemical treatment or protein engineering of antibodies is required for incorporation of the bioorthogonal handles, processes that often affect antibody folding, stability, and/or production yield and cost. Accordingly, concurrent with advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in site-specifically labeling native (nonengineered) antibodies without chemical or enzymatic treatments. In this review, we highlight recent strategies for producing site-specific native antibody conjugates and provide a comprehensive summary of the merits and disadvantages of these strategies.

Diselenide crosslinks for enhanced and simplified oxidative protein folding

The in vitro oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin’s folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, limiting its folding rate and yield in vitro. Aiming to overcome these limitations, we undertake systematic investigation of diselenide bridges at native and non-native positions and investigate their effect on hirudin’s folding, structure and activity. Our studies demonstrate that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogues, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirms that the diselenide substitutions maintained the overall three-dimensional structure of the protein and left its function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

Folding of Truncated Granulin Peptides.

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a “mini-granulin” structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) ~20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm−1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

STUDY OF PHYSICAL AND CHEMICAL INDICATORS OF DAIRY PRODUCTS ON THE BASIS OF THE MAXIREN ENZYME AND PROBIOTIC PREPARATION «NARINE»

The article presents the results of experimental studies of the quality indicators of milk and dairy products (rennet cheese, yogurt), prepared using the enzyme preparation «Maxiren» and the probiotic preparation «Narіne». The preparation «Maxiren 1800 granulate» has the following composition: special dairy yeast Kluyveromyces marxianus and chymosin 50%, pepsin 50% and are made from calves, lambs, goats and beef pepsin. Starter direct introduction TM GOOD FOOD «Narine» contains a complex of associated microorganisms Streptococcus salivariussub, Lactobacillus acidophilus, Lactobacillus delbrueckiisub.The determination of the organoleptic and physicochemical indicators of the quality of milk and products based on it was carried out according to generally accepted methods. In the organoleptic evaluation of milk and lactic acid products, the color of the milk, smell, taste, texture, visible purity, the presence of impurities, mold stains and phase separation were investigated. Determination of active acidity was carried out according to GOST 26781. Determination of acidity in degrees Turner was performed according to GOST 3624. The percentage of fat, protein, skimmed milk residue (SOMO), the amount of water introduced, that is, the falsification of milk, and the density was determined using an ultrasonic milk analyzer ECOMILK-M.It was revealed that the acidity of the lactic acid product on the basis of the «Narine» – 850T, active – 4.7. The product has a pure sour-milk taste, a dense and rather viscous consistency that does not deteriorate during storage.It has been established that the Maxiren enzyme does not ferment protein to short peptides that cause bitterness in cheese, has optimal milk-clotting properties, stable taste indicators, ensuring maximum milk folding and product yield. The product has a pure sour milk, moderately salty taste, no foreign tastes and odors, milky white color.

Impact of bacterial chaperonin GroEL\u2013GroES on bacteriorhodopsin folding and membrane integration

Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV–Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL–GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.

Backbone cyclization of analgesic conotoxin gexiva facilitates direct folding of the ribbon isomer

Conotoxin GeXIVA inhibits the α9α10 nicotinic acetylcholine receptor (nAChR) and is analgesic in animal models of pain. α-Conotoxins have four cysteines that can have three possible disulfide connectivities: globular (CysI–CysIII and CysII–CysIV), ribbon (CysI–CysIV and CysII–CysIII), or bead (CysI–CysII and CysIII–CysIV). Native α-conotoxins preferably adopt the globular connectivity, and previous studies of α-conotoxins have focused on the globular isomers as the ribbon and bead isomers typically have lower potency at nAChRs than the globular form. A recent report showed that the bead and ribbon isomers of GeXIVA are more potent than the globular isomer, with low nanomolar half-maximal inhibitory concentrations (IC50). Despite this high potency, the therapeutic potential of GeXIVA is limited, because like most peptides, it is susceptible to proteolytic degradation and is challenging to synthesize in high yield. Here we used backbone cyclization as a strategy to improve the folding yield as well as increase the serum stability of ribbon GeXIVA while preserving activity at the α9α10 nAChR. Specifically, cyclization of ribbon GeXIVA with a two-residue linker maintained the biological activity at the human α9α10 nAChR and improved stability in human serum. Short linkers led to selective formation of the ribbon disulfide isomer without requiring orthogonal protection. Overall, this study highlights the value of backbone cyclization in directing folding, improving yields, and stabilizing conotoxins with therapeutic potential.

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

Structural Variants of a Liver Fluke Derived Granulin Peptide Potently Stimulate Wound Healing.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.

Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System

Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted tothe early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.

Folding and stabilizing membrane proteins in amphipol A8-35.

Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called 'amphipols' (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both α-helical and β-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.

Liprotides assist in folding of outer membrane proteins.

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α-lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α-lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non-ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8-9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding-inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.

The folding, stability and function of lactose permease differ in their dependence on bilayer lipid composition

Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different membrane protein properties have the same dependence on membrane mechanics, or whether mechanics are tuned to specific protein processes of the protein. We study the influence of lipid lateral pressure and electrostatic effects on the in vitro reconstitution, folding, stability and function of a representative of the ubiquitous major facilitator transporter superfamily, lactose permease. Increasing the outward chain lateral pressure in the bilayer, through addition of lamellar phosphatidylethanolamine lipids, lowers lactose permease folding and reconstitution yields but stabilises the folded state. The presence of phosphatidylethanolamine is however required for correct folding and function. An increase in headgroup negative charge through the addition of phosphatidylglycerol lipids favours protein reconstitution but is detrimental to topology and function. Overall the in vitro folding, reconstitution, topology, stability and function of lactose permease are found to have different dependences on bilayer composition. A regime of lipid composition is found where all properties are favoured, even if suboptimal. This lays ground rules for rational control of membrane proteins in nanotechnology and synthetic biology by manipulating global bilayer properties to tune membrane protein behaviour.

Backbone cyclization of analgesic conotoxin GeXIVA facilitates direct folding of the ribbon isomer

Conotoxin GeXIVA inhibits the α9α10 nicotinic acetylcholine receptor (nAChR) and is analgesic in animal models of pain. α-Conotoxins have four cysteines that can have three possible disulfide connectivities: globular (CysI-CysIII and CysII-CysIV), ribbon (CysI-CysIV and CysII-CysIII), or bead (CysI-CysII and CysIII-CysIV). Native α-conotoxins preferably adopt the globular connectivity, and previous studies of α-conotoxins have focused on the globular isomers as the ribbon and bead isomers typically have lower potency at nAChRs than the globular form. A recent report showed that the bead and ribbon isomers of GeXIVA are more potent than the globular isomer, with low nanomolar half-maximal inhibitory concentrations (IC50). Despite this high potency, the therapeutic potential of GeXIVA is limited, because like most peptides, it is susceptible to proteolytic degradation and is challenging to synthesize in high yield. Here we used backbone cyclization as a strategy to improve the folding yield as well as increase the serum stability of ribbon GeXIVA while preserving activity at the α9α10 nAChR. Specifically, cyclization of ribbon GeXIVA with a two-residue linker maintained the biological activity at the human α9α10 nAChR and improved stability in human serum. Short linkers led to selective formation of the ribbon disulfide isomer without requiring orthogonal protection. Overall, this study highlights the value of backbone cyclization in directing folding, improving yields, and stabilizing conotoxins with therapeutic potential.