Small Amino Acids Research Articles

Combined Toxicity of Multi-Walled Carbon Nanotubes and Cu2+ on the Growth of Ryegrass: Effect of Surface Modification, Dose, and Exposure Time Pattern

The escalating release of multi-walled carbon nanotubes (MWCNTs) into the environment has raised concerns due to their potential ecotoxicological impacts. However, their combined phytotoxicity with heavy metals such as copper (Cu) is still unclear. This study investigated the individual and combined toxic effects of MWCNTs (MWCNT, MWCNT-OH, and MWCNT-COOH) and Cu2+ on ryegrass (Lolium multiflorum), uniquely considering different addition orders. The results show that Cu severely inhibited the growth of ryegrass while MWCNTs exhibited a hormesis effect on ryegrass. When MWCNT and Cu were combined, the malondialdehyde (MDA) content in ryegrass showed a 32.39% increase at 20 mg/L MWCNT exposure, suggesting reduced oxidative stress. However, at the higher concentration of 1000 mg/L, it led to a significant 75.22% reduction in ryegrass biomass. MWCNT-COOH had the most pronounced effect, reducing the total chlorophyll content by 39.76% compared to unmodified MWCNT and by 10.67% compared to MWCNT-OH (500 mg/L). Additionally, pre-induced MWCNTs might alleviate the Cu in the plant by 23.08–35.38% through adsorption in the nutrient solution. Small molecule organic acids and amino acids primarily mediated the response to environmental stress in ryegrass. This research provides crucial insights into understanding the complex interactions of MWCNT and Cu2+ and their combined effects on plant ecosystems.

Scalar and vectorized implementations of the density functional theory via Geant4 and GeantV project

In this paper, we present the implementation of the Density Functional Theory (DFT) method using the Geant4-DNA framework in the Single Instruction Single Data (SISD) mode. Furthermore, this implementation is improved in terms of execution time within the GeantV project with vectorization techniques such as Single Instruction Multiple Data (SIMD). Within this framework, a set of SIMD strategies in molecular calculation algorithms such as one-electron operators, two-electron operator, quadrature grids, and functionals, was implemented using the VecCore library. The applications developed in this work implement two DFT functionals, the Local Density Approximation (LDA) and the General Gradient Approximation (GGA), to approximate the molecular ground-state energies of small molecules and amino acids. To assess the performance of the implementations, a standard test simulation was performed in multiple CPU platforms. The SIMD vectorization strategy significantly accelerates DFT calculations, leading to time ratios ranging from 1.6 to 5.4 in either individual steps or entire implementations when compared with the scalar process within Geant4.

Derivatization of Microcystins Can Increase Target Inhibition while Reducing Cellular Uptake.

Microcystins, a large family of nonribosomal cyclic heptapeptides known for their hepatotoxicity, are among the best-studied cyanobacterial toxins. Recently, they have been discussed as leads for the development of anticancer drug substances. Their main mode-of-action is inhibition of the eukaryotic serine/threonine protein phosphatases 1 and 2A. Unlike many cytotoxins that can cross cell membranes by passive diffusion, microcystins depend on active uptake via organic anion transporting polypeptides 1B1 or 1B3. Both phosphatase inhibition and transportability strongly depend on the structure of the individual microcystin. Here, we present how chemical modification of positions 2 and 4 of the microcystin core structure can alter these two properties. Aiming to reduce transportability and increase phosphatase inhibition, we used pharmacophore modeling to investigate the phosphatase inhibition potential of microcystins derivatized with small molecules containing a variety of functional groups. The respective derivatives were synthesized using click chemistry. We discovered that some derivatized microcystins can address a yet undescribed subpocket of the protein phosphatase 1. The derivatized microcystins were tested for phosphatase 1 inhibition and cytotoxicity on transporter-expressing cell lines, revealing that target inhibition and transportability of microcystins can independently be influenced by the physicochemical properties, especially of the residue located in position 2 of the microcystin. Derivatization with small acids or amino acids resulted in microcystins with a favorable ratio of inhibition to transportability, making these derivatives potentially suitable for drug development.

Underlying mechanisms of different polysaccharides on the texture characteristics and protein digestibility of soybean curd

This study aimed to investigate the effects of the addition of potato starch (PS) and guar gum (GG) at different concentrations (0.4 and 0.8%) on the physical properties, protein conformation, and in vitro digestibility of soybean curd. The results indicated that the PS acted as a filler in the protein structure, enhancing the firmness, springiness, and chewiness of the soybean curd. GG encapsulated the disorganized filamentous protein matrix, increasing the adhesiveness of the soybean curd. The addition of GG and PS reduced the hydrophobic interactions and disulfide bonds responsible for cross-linking soybean curd proteins, while forming more hydrogen bonds with the proteins (p < 0.05). GG formed the most hydrogen bonds with the proteins, as well as reduced β-sheet content and transformed into more random coil and β-turn (p < 0.05). Also, the addition of GG (particularly at 0.8%) significantly reduced the degree of protein hydrolysis (31.98%) in soybean curd, resulting in fewer small molecular weight peptides and lower total amino acid content (3386.79 μg/g protein) at the end of in vitro digestion (p < 0.05). In contrast, the addition of a small amount of PS (0.4%) did not affect the protein digestibility of soybean curd. This study clarified the impact of polysaccharide additions on modifying the soybean curd texture and protein digestibility.

Unlocking the roles of wheat root exudates in regulating laccase-catalyzed estrogen humification

While laccase humification has an efficient capacity to convert estrogenic pollutants, the roles of wheat (Triticum aestivum L.) root exudates (W-REs) in the enzymatic humification remain poorly understood. Herein, we presented the research into the effects of W-REs on 17β-estradiol (E2) and bisphenol A (BPA) conversion in vitro laccase humification. W-REs inhibited E2 removal but promoted BPA conversion in the enzymatic humification, and the first-order kinetic constants for E2 and BPA were 0.27–0.69 and 0.28–0.55 h−1, respectively. Specialized small phenols and amino acids in W-REs were susceptible to laccase humification, resulting in increased copolymerization of estrogen and W-REs. In greenhouse hydroponics, the accumulated amounts of E2 (BPA) in the roots and shoots were estimated to be 0.87 (2.15) and 0.43 (0.51) nmol·plant−1 at day 3, respectively. By forming low- and eventually non-toxic copolymeric precipitates between estrogen and W-REs, laccase humification lowered the phytotoxicity and bioavailability of estrogen in the rhizosphere solution, consequently relieving its uptake, accumulation, and distribution in the wheat cells. This work sheds light on the roles of W-REs in regulating laccase-catalyzed estrogen humification, and gives an insight into the path of addressing organic contamination in the rhizosphere and ensuring food safety.

A comparative study of structural and digestive properties of bovine, goat and human α-Lactalbumin with different calcium binding levels

α-Lactalbumin (α-LA), the primary whey protein in human milk, is rich in essential amino acids and plays a crucial role in infant growth and development. This study investigated the effects of different calcium (Ca) binding levels on the structural characteristics and gastrointestinal digestive behavior of bovine, goat and human α-LA. We used spectroscopic techniques to detect changes in the secondary and tertiary structure of α-LA and analyzed thermal stability with differential scanning calorimetry. Decalcification led to partial unfolding of α-LA, resulting in a more open conformation and significantly reduced thermal stability. Additionally, decalcification increased the in vitro digestibility of α-LA under the infant digestion condition, diminishing its resistance to pepsin and yielding more small peptides and free amino acids. Conversely, higher Ca content slightly affected the structure of α-LA and reduced its digestibility. Bovine, goat and human α-LA exhibited differences in structure and digestion patterns. Human α-LA showed the lowest gastric digestibility, with hydrolysis occurring mainly on large peptides rather than intact proteins. However, during intestinal digestion, human α-LA demonstrated higher digestibility than bovine and goat α-LA, releasing more small peptides and amino acids for more complete digestion. These results provide insights into the relationship between the structure and digestibility of α-LA, which can inform the formulation of infant formulas aimed at optimizing protein digestion and nutrient absorption.

Deciphering Structural Traits for Thermal and Kinetic Stability Across Protein Family Evolution through Ancestral Sequence Reconstruction.

Natural proteins are frequently marginally stable, and an increase in environmental temperature can easily lead to unfolding. As a result, protein engineering to improve protein stability is an area of intensive research. Nonetheless, since there is usually a high degree of structural homology between proteins from thermophilic organisms and their mesophilic counterparts, the identification of structural determinants for thermo-adaptation is challenging. Moreover, in many cases, it has become clear that the success of stabilization strategies is often dependent on the evolutionary history of a protein family. In the last few years, the use of ancestral sequence reconstruction (ASR) as a tool for elucidation of the evolutionary history of functional traits of a protein family have gained strength. Here, we used ASR to trace the evolutionary pathways between mesophilic and thermophilic kinases that participate in the biosynthetic pathway of vitamin B1 in bacteria. By combining biophysics approaches, X-ray crystallography, and molecular dynamic simulations, we found that the thermal stability of these enzymes correlate with their kinetic stability, where the highest thermal/kinetic stability is given by an increase in small hydrophobic amino acids that allow a higher number of interatomic hydrophobic contacts, making this type of interaction the main support for stability in this protein architecture. The results highlight the potential benefits of using ASR to explore the evolutionary history of protein sequence and structure to identify traits responsible for the kinetic and thermal stability of any protein architecture.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Safety of soy leghemoglobin from genetically modified Komagataella phaffii as a food additive.

The EFSA Panel on Food Additive and Flavourings (FAF Panel) provides a scientific opinion on the safety of soy leghemoglobin from genetically modified Komagataella phaffii as a food additive in accordance with Regulation (EC) No 1331/2008. The proposed food additive, LegH Prep, is intended to be used as a colour in meat analogue products. The yeast Komagataella phaffii strain MXY0541 has been genetically modified to produce soy leghemoglobin; the safety of the genetic modification is under assessment by the EFSA GMO Panel (EFSA-GMO-NL-2019-162). The amount of haem iron provided by soy leghemoglobin from its proposed uses in meat analogue products is comparable to that provided by similar amounts of different types of meat. The exposure to iron from the proposed food additive, both at the mean and 95th percentile exposure, will be below the 'safe levels of intake' established by the NDA Panel for all population groups. Considering that the components of the proposed food additive will be digested to small peptide, amino acids and haem B; the recipient (non GM) strain qualifies for qualified presumption of safety status; no genotoxicity concern has been identified and no adverse effects have been identified at the highest dose tested in the available toxicological studies, the Panel concluded that there was no need to set a numerical acceptable daily intake (ADI) and that the food additive does not raise a safety concern at the proposed use in food category 12.9 and maximum use level. The Panel concluded that the use of soy leghemoglobin from genetically modified Komagataella phaffii MXY0541 as a new food additive does not raise a safety concern at the proposed use and use level. This safety evaluation of the proposed food additive remains provisional subject to the ongoing safety assessment of the genetic modification of the production strain by the GMO Panel (EFSA-GMO-NL-2019-162).

Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2

ASCT2 (alanine serine cysteine transporter 2), a member of the SLC1 (solute carrier 1) family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anti-cancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor Lc-BPE with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that, despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The pre-incubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

A multi-hierarchical approach reveals d-serine as a hidden substrate of sodium-coupled monocarboxylate transporters

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

Cryo-EM structure of the human Asc-1 transporter complex

The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.

Enhancing the nutritional and bioactive benefits of faba bean flour by combining preprocessing and thermoplastic extrusion: A comprehensive study on digestion-resistant peptides

This research assessed how three preprocessing techniques [soaking (S), soaking and reconstitution (SR), and soaking and dehulling (SD)] impact the protein digestibility and bioactivity of faba bean flours when combined with thermoplastic extrusion. Samples were compared against a control (C) of extruded faba bean flour without preprocessing. Applying preprocessing techniques followed by extrusion diminished antinutrient levels while enhancing protein hydrolysis and in vitro bioactivity in higher extent compared to C. Specifically, SD combined with extrusion was the most effective, achieving an 80% rate of protein hydrolysis and uniquely promoting the release of gastric digestion-resistant proteins (50-70 kDa). It also resulted in the highest release of small peptides (<3kDa, 22.51%) and free amino acids (15.50%) during intestinal digestion. Moreover, while all preprocessing techniques increased antioxidant (ABTS radical-scavenging), antidiabetic, and anti-hypertensive activities, SD extruded flour displayed the highest levels of dipeptidyl peptidase inhibition (DPP-IVi, IC50=13.20 µg/mL), pancreatic α-amylase inhibition (IC50=8.59 mg/mL), and angiotensin I-converting enzyme inhibition (ACEi, IC50=1.71 mg protein/mL). As a result, it was selected for further peptide and in silico bioactive analysis. A total of 24 bioactive peptides were identified in intestinal digests from SD extruded flour, all with potential DPP-IVi and ACEi activities, and six were also predicted as antioxidant peptides. VIPAGYPVAIK and GLTETWNPNHPEL were highlighted as resistant bioactive peptides with the highest antidiabetic and antioxidant potential. Our findings demonstrated that combining preprocessing (particularly SD) and thermoplastic extrusion enhances protein digestibility in faba beans and promotes the release of beneficial bioactive peptides in the intestine.

Bacterial over-production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands

BackgroundSLC38A2 is a ubiquitously expressed Na+-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy.Methods and ResultsThe cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane.ConclusionsThe addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.

The Digestive Vacuole of the Malaria Parasite: A Specialized Lysosome.

The malaria parasite resides within erythrocytes during one stage of its life cycle. During this intraerythrocytic period, the parasite ingests the erythrocyte cytoplasm and digests approximately two-thirds of the host cell hemoglobin. This digestion occurs within a lysosome-like organelle called the digestive vacuole. Several proteases are localized to the digestive vacuole and these proteases sequentially breakdown hemoglobin into small peptides, dipeptides, and amino acids. The peptides are exported into the host cytoplasm via the chloroquine-resistance transporter and an amino acid transporter has also been identified on the digestive vacuole membrane. The environment of the digestive vacuole also provides appropriate conditions for the biocrystallization of toxic heme into non-toxic hemozoin by a poorly understood process. Hemozoin formation is an attribute of Plasmodium and Haemoproteus and is not exhibited by other intraerythrocytic protozoan parasites. The efficient degradation of hemoglobin and detoxification of heme likely plays a major role in the high level of replication exhibited by malaria parasites within erythrocytes. Unique features of the digestive vacuole and the critical importance of nutrient acquisition provide therapeutic targets for the treatment of malaria.

Combined functional genomic and metabolomic approaches identify new genes required for growth in human urine by multidrug-resistant Escherichia coli ST131.

Uropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance.

Engineering of Candida antarctica Lipase B for Enhanced Perhydrolysis Activity by Modulating the Potential Water Channel

AbstractAlthough Candida antarctica lipase B (CAL‐B) naturally hydrolyzes an ester compounds, it can catalyze a variety of promiscuous reactions, such as perhydrolysis, aldol reaction, and Michael‐type reactions. Perhydrolysis is similar to hydrolysis but uses hydrogen peroxide instead of water molecules. It was recently proposed that water molecules access the active site of CAL‐B through a water channel. We hypothesized that hydrogen peroxide may also use the channel to enter the active site, and that the access of hydrogen peroxide is one of the key factors for perhydrolysis activity. We investigated the effects of mutations at two residues in the water channel of CAL‐B on the perhydrolysis activity. The residues, Pro280 and Ala281, are located at the constricted area of the water channel. We found that the substitution of Ala281 to a small and polar amino acid, such as serine or threonine, resulted in a noticeable increase in hydrolysis and perhydrolysis activity. The activity of the variant enzymes was up to three times and twice higher for the activities of hydrolysis and perhydrolysis, respectively, than those of CAL‐B. In contrast, the substitution of the Pro280 residue with a small and polar amino acid decreased both hydrolysis and perhydrolysis activities.

Encoding Structure in Intrinsically Disordered Protein Biomaterials.

In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs. Nature also diversifies the structure of proteins and thereby their functions by hybridization of the proteins with other moieties such as glycans and lipids; for instance, post-translationally glycosylated and lipidated proteins are important cell membrane components. Additionally, diversity in protein structure and function is achieved in nature through cross-linking proteins within themselves or with other domains to create various topologies. For example, an essential characteristic of the extracellular matrix (ECM) is the cross-linking of its network components, including proteins such as collagen and elastin, as well as polysaccharides such as hyaluronic acid (HA). Inspired by nature, synthetic IDP (SynIDP)-based biomaterials can be designed by employing similar strategies with the goal of introducing structural diversity and hence unique physiochemical properties. This Account describes such materials produced over the past decade and following one or more of the following approaches: (1) incorporating highly ordered domains into SynIDPs, (2) conjugating SynIDPs to other moieties through either genetically encoded post-translational modification or chemical conjugation, and (3) engineering the topology of SynIDPs via chemical modification. These approaches introduce modifications to the primary structure of SynIDPs, which are then translated to unique three-dimensional secondary and tertiary structures. Beginning with completely disordered SynIDPs as the point of origin, structure may be introduced into SynIDPs by each of these three unique approaches individually along orthogonal axes or by combinations of the three, enabling bioinspired designs to theoretically span the entire range of three-dimensional structural possibilities. Furthermore, the resultant structures span a wide range of length scales, from nano- to meso- to micro- and even macrostructures. In this Account, emphasis is placed on the physiochemical properties and structural features of the described materials. Conjugates of SynIDPs to synthetic polymers and materials achieved by simple mixing of components are outside the scope of this Account. Related biomedical applications are described briefly. Finally, we note future directions for the design of functional SynIDP-based biomaterials.

Production and characterization of homologous protoporphyrinogen IX oxidase (PPO) proteins: Evidence that small N-terminal amino acid changes do not impact protein function.

Transgenic soybean, cotton, and maize tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides have been developed by introduction of a bacterial-derived PPO targeted into the chloroplast. PPO is a membrane-associated protein with an intrinsic tendency for aggregation, making expression, purification, and formulation at high concentrations difficult. In this study, transgenic PPO expressed in three crops was demonstrated to exhibit up to a 13 amino acid sequence difference in the N-terminus due to differential processing of the chloroplast transit peptide (CTP). Five PPO protein variants were produced in and purified from E. coli, each displaying equivalent immunoreactivity and functional activity, with values ranging from 193 to 266 nmol min-1 mg-1. Inclusion of an N-terminal 6xHis-tag or differential processing of the CTP peptide does not impact PPO functional activity. Additionally, structural modeling by Alphafold, ESMfold, and Openfold indicates that these short N-terminal extensions are disordered and predicted to not interfere with the mature PPO structure. These results support the view that safety studies on PPO from various crops can be performed from a single representative variant. Herein, we report a novel and robust method for large-scale production of PPO, enabling rapid production of more than 200 g of highly active PPO protein at 99% purity and low endotoxin contamination. We also present a formulation that allows for concentration of active PPO to > 75 mg/mL in a buffer suitable for mammalian toxicity studies.

Imaging specific proteins in living cells with small unnatural amino acid attached Raman reporters.

Fluorescence labeling via fluorescent proteins (FPs) or immunofluorescence has been routinely applied for microscopic imaging of specific proteins. However, due to these over-weight and oversized labels (e.g. GFP, 238 aa, 27 kDa, ∼4 nm in size), the potential physiological malfunctions of the target proteins are largely underestimated in living cells. Herein, for living cells, we report a small and minimally-invasive Raman reporter (about 2 aa and <1 kDa), which can be site-specifically introduced into proteins by genetic codon expansion. After a single unnatural amino acid (UAA) is precisely incorporated into the target protein, the strained alkyne can rapidly undergo copper-free Diels-Alder cycloaddition reactions with the tetrazine-functionalized Raman reporter, which features a fine vibrational spectrum in contrast to fluorescence. In our experimental results, the UAA-based Raman tag was successfully incorporated into vimentin, histone 3.3 and huntingtin (Htt74Q) proteins in living HeLa cells and further utilized for stimulated Raman imaging. The site-specific bioorthogonal fusion of small Raman tags with intracellular proteins will pave the way for minimally-invasive protein labeling and multi-color imaging in living cells.