Native Protein Research Articles

Mapping of β3-Adrenergic Receptor in Living Cells with a Ligand-Guided Fluorescent Probe.

Tracking the dynamic distribution of native proteins in living cells or tissues in real time is essential for understanding the functional mechanisms involved in their physiological or pathological processes. The β3-adrenergic receptor (β3-AR) has important biological functions. It is expected to be a diagnostic indicator for aging, yet current probes for β-ARs are unable to dynamically monitor β3-AR in real time, which impedes the progress of β3-AR research. Here, we developed a ligand-directed anchoring probe, β3-ARP, that precisely covalently anchors with native β3-AR in living cells, for the diagnosis of senescence. The ligand-directed anchoring probe selectively recognizes and traces β3-AR and can achieve dynamic observation and monitoring of β3-AR in living cells, including the interaction between lipid droplets and mitochondria. Moreover, we were able to directly probe the distribution of β3-AR in various organs of aging mice in situ and track the location of native β3-AR in different types of primary neuronal cells using β3-ARP and two-photon imaging, which revealed the aberrant accumulation of lipid droplets and the distribution of β3-AR in neurological diseases. This research has resulted in a new covalently anchoring fluorescent probe, β3-ARP, which could serve as a powerful tool to explore the link between β3-AR and age-related diseases.

Antibacterial and cytotoxic properties of esterified α- lactalbumin and β-lactoglobulin

α -Lactalbumin ( α -La) or β -lactoglobulin ( β -Lg) were esterified with methanol. Native proteins and their methylated esters (Est. α -La and Est. β -Lg) were tested for their antibacterial and cytotoxic properties. Esterification extents for Est. α -La and Est. β -Lg were 92% and 88%, with isoelectric points at pH 8 and 10, respectively. The Est. α -La and Est. β -Lg were more inhibiting the growth of pathogenic bacteria compared to the corresponding native proteins. The impact of esterified α -La and β -Lg on MCF-7, A549, and Caco-2 cell lines was assessed and revealed that the concentration inhibiting 50% cellular growth (IC50) for Est. α -La recorded 107.93 µg/mL against MCF-7, 616.44 µg/mL against A-549, and 388.23 µg/mL against Caco2. In parallel, Est. β -Lg achieved IC50 at 101.35, 420.84, and 234.16 µg/mL, against MCF-7, A-549, and Caco2, respectively. Esterified proteins may serve as therapeutic antimicrobial agents against pathogenic bacteria and cytotoxic agents against specific cancer cells.

Membrane Fusion Mediated by Cationic Helical Peptide L-MMBen through Phosphatidylglycerol Recruitment.

Membrane fusion is the basis for many biological processes, which holds promise in biomedical applications including the creation of engineered hybrid cells and cell membrane functionalization. Extensive research efforts, including investigations into DNA zippers and carbon nanotubes, have been dedicated to the development of membrane fusion strategies inspired by natural SNARE proteins; nevertheless, achieving a delicate balance between membrane selectivity and high fusion efficiency through precise molecular engineering remains unclear. In our recent study, we successfully designed L-MMBen, a cationic helical antimicrobial peptide that exhibits remarkable antimicrobial efficacy while demonstrating moderate cytotoxicity. In this work, we demonstrate the effective and selective induction of fusion between phosphatidylglycerol (PG)-containing membranes by L-MMBen. By combining biophysical assays at the single-vesicle level with computer simulations at the molecular level, we discovered that L-MMBen can stably adsorb onto the surface of PG-containing membranes, leading to the formation of stalk structures between vesicles and ultimately resulting in membrane fusion. Furthermore, the occurrence of fusion is attributed to the unique ability of L-MMBen to recruit PG lipids and bridge adjacent vesicles. In contrast, its nonhelical counterpart DL-MMBen was found to lack this capability despite possessing an identical positive charge. These findings present an alternative molecule for achieving selective membrane fusion and provide insights for designing helical peptides with diverse applications.

Cytochromes b5 Occurrence in Viruses Belonging to the Order Megavirales.

Cytochrome b5 is a small electron transport protein that is found in animals, plants, fungi and photosynthetic proteobacteria where it plays key metabolic roles in energy production, lipid and sterol biosynthesis and cytochrome P450 biochemistry. Previously it was shown that a gene encoding a soluble and functional cytochrome b5 protein was encoded in the large double stranded DNA virus OtV2 that infects the unicellular marine green alga Ostreococcus tauri, the smallest free-living eukaryote described to-date. This single gene represented a unique finding in the virosphere. We now report that genes for soluble and membrane-bound cytochromes b5 also occur in giant viruses in the proposed order Megavirales, particularly the AT-rich Mimiviridae and Tupanviruses. Conversely, other members of the Megaviralestaxa such as the GC-rich Pandoraviridae have not been found to encode cytochrome b5 as yet. Megaviruses encoding cytochrome b5 have been isolated from the deep ocean, from freshwater and terrestrial sources, as well as from human patients. Giant virus cytochrome b5 proteins share high sequence identity with one another (45-95% depending on group) but no more than 25% identity with the cytochrome b5 gene product we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique cytochrome b5 genes in giant viruses remainsunknown. Examination of viral cytochrome b5 primary amino acid sequences revealed that some have either a N- or C-terminal transmembrane anchor, whilst others lack a membrane anchor and are thus predicted to be soluble proteins. This cytochrome b5 topography suggests adapted biochemical functions in those viruses. Our findings raise questions regarding the evolution and diversity of cytochrome b5 proteins in nature, adding to questions about the origin of viral haemoproteins in general.

Engineering GID4 for use as an N-terminal proline binder via directed evolution.

Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.

Allergenic Potential of Common Hops (Humulus lupulus L.) in the Context of Cross-Reactions with Pollen Allergens.

Common hops (Humulus lupulus L.) play a key role in brewing, providing the bitterness, flavor, and aroma of beer, and are widely used in supplements for their antibacterial, anti-inflammatory, and antioxidant properties. However, despite their broad applications, the allergenic potential of common hops remains underexplored, particularly when compared to the closely related Humulus japonicus. This preliminary study aimed to investigate the allergenic potential of common hops and their potential cross-reactivity with common pollen allergens. The immunoreactivity of hop stalks, leaves, and cones was assessed using antibodies against major allergens from birch (Bet v1a), mugwort (Art v1), and timothy grass (Phl p5b), as well as three sera from pollen-allergic patients. Slot Blot analysis was performed using phosphate-buffered saline extracts from the stalks, leaves, and cones of three hop cultivars, while Western Blotting followed SDS-PAGE protein separation. The results revealed significant immunoreactivity in native hop proteins, with diminished reactivity observed in denatured proteins. Cross-reactivity between hop proteins and major pollen allergens was confirmed, indicating that hop proteins may contribute to allergic sensitization in pollen-sensitive individuals. These findings underscore the potential allergenic risks associated with the consumption or exposure to hop-containing products.

Single-cell chemoproteomics identifies metastatic activity signatures in breast cancer.

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

Comparative analysis of purity of human albumin preparations for clinical use

BackgroundAlbumin is the most prevalent plasma protein and serves numerous physiological roles, both in body fluid management and in various other capacities. In many diseases, a deficiency of albumin has been observed, and in certain conditions, albumin substitution has been demonstrated to improve outcome in comparison to plasma expansion using crystalloid or other colloid solutions. The favourable effects of using albumin in patients with liver cirrhosis are likely associated with the non-oncotic functions of albumin. Albumin for clinical use is obtained through fractionation of pooled donor plasma. The production procedures are optimized to ensure pure, chemically uncompromised and native protein. ResultsWe have extensively analysed commercial preparations of human albumin for clinical use from six different providers. Parameters that must correspond to the requirements of international pharmacopoeias were assessed (aluminium, ethanol, sodium, the presence of dimers and oligomers) and found to conform in all cases. In addition, we used for the first time nuclear magnetic resonance (NMR) as an additional analytical approach for investigating in greater depth the quality of a biological remedy gained from human plasma. We applied both 1H NMR and 13C-HSQC for confirming the identity of the albumin preparations, which also conformed in all cases. Moreover, we utilized T2-filtered 1H NMR and 13C-HSQC measurements to identify the presence of small molecules in the preparations. This demonstrated similar patterns of additional substances present, but also unveiled certain differences in purity in the products of the different providers. SignificanceOur analyses confirmed that albumin preparations in clinical use conform to the requirements. We furthermore demonstrate that NMR measurements can provide further depth in identity and purity measurements of biologicals. Despite largely standardized protocols in pharmaceutical albumin production, our in-depth analyses revealed differences in purity. Some samples exhibited lower levels of components other than albumin. We discuss possible causes of these observations and their potential implications for clinical therapy.

Evaluation of protective efficacy of recombinant Toxoplasma gondii DDX39 protein vaccine against acute and chronic T. gondii infection in mice

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+T cells rising by 21.9% and CD8+T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response.Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15 to 20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

Recent Advances in Artificial Anion Channels and Their Selectivity.

Nature performs critical physiological functions using a series of structurally and functionally diverse membrane proteins embedded in cell membranes, in which native ion protein channels modify the electrical potential inside and outside the cell membrane through charged ion movements. Consequently, the cell responds to external stimuli, playing an essential role in various life activities, such as nerve excitation conduction, neurotransmitter release, muscle movement, and control of cell differentiation. Supramolecular artificial channels, which mimic native protein channels in structure and function, adopt unimolecular or self-assembled structures, such as crown ethers, cyclodextrins, cucurbiturils, column arenes, cyclic peptide nanotubes, and metal-organic artificial channels, in channel construction strategies. Owing to the various driving forces involved, artificial synthetic ion channels can be divided into artificial cation and anion channels in terms of ion selectivity. Cation selectivity usually originates from ion coordination, whereas anion selectivity is related to hydrogen bonding, ion pairing, and anion-dipole interactions. Several studies have been conducted on artificial cation channels, and several reviews have summarized them in detail; however, the research on anions is still in the initial stages, and related reviews have rarely been reported. Hence, this article primarily focuses on the recent research on anion channels.

Engineered protein destabilization reverses intrinsic immune evasion for candidate vaccine pan-strain KSHV and SARS-CoV-2 antigens.

Both Kaposi sarcoma herpesvirus LANA and SARS coronavirus 2 RdRp/nsp12 are highly conserved replication proteins that evade immune processing. By deleting the LANA central repeat 1 domain (LANA ΔCR1 ) or by dividing RdRp into two separated fragments (RdRp Frag ) to maximize nascent protein mis-folding, cis peptide presentation was increased. Native LANA or RdRp SIINFEKL fusion proteins expressed in MC38 cancer cells were not recognized by activated OT-1 CD8 + cells against SIINFEKL but cytotoxic recognition was restored by expression of the corresponding modified proteins. Immunocompetent syngeneic mice injected with LANA- or RdRp-SIINFEKL MC38 cells developed rapidly-growing tumors with short median survival times. Mice injected with LANA ΔCR1 - or RdRp Frag -SIINFEKL had partial tumor regression, slower tumor growth, longer median survival, as well as increased effector-specific tumor-infiltrating lymphocytes. These mice developed robust T cell responses lasting at least 90 days post-injection that recognized native viral protein epitopes. Engineered vaccine candidate antigens can unmask virus-specific CTL responses that are typically suppressed during native viral infection.

Origin of Pressure Resistance in Deep-Sea Lactate Dehydrogenase.

High hydrostatic pressure has a dramatic effect on biochemical systems, as exposure to high pressure can result in structural perturbations ranging from dissociation of protein complexes to complete denaturation. The deep ocean presents an interesting paradox since it is teeming with life despite the high-pressure environment. This is due to evolutionary adaptations in deep-sea organisms, such as amino acid substitutions in their proteins, which aid in resisting the denaturing effects of pressure. However, the physicochemical mechanism by which these substitutions can induce pressure resistance remains unknown. Here, we use molecular dynamics simulations to study pressure-adapted lactate dehydrogenase from the deep-sea abyssal grenadier (Coryphaenoides armatus), in comparison with that of the shallow-water Atlantic cod (Gadus morhua). We examined structural, thermodynamic and volumetric contributions to pressure resistance, and report that the amino acid substitutions result in a decrease in volume of the deep-sea protein accompanied by a decrease in thermodynamic stability of the native protein. Our simulations at high pressure also suggest that differences in compressibility may be important for understanding pressure resistance in deep-sea proteins.

Recruitment to the Proteasome Is Necessary but Not Sufficient for Chemically Induced, Ubiquitin-Independent Degradation of Native Proteins.

Targeted protein degradation (TPD) is a promising strategy for drug development. Most degraders function by forcing the association of the target protein (TP) with an E3 Ubiquitin (Ub) ligase, which, in favorable cases, results in the polyubiquitylation of the TP and its subsequent degradation by the 26S proteasome. An alternative strategy would be to create chemical dimerizers that bypass the requirement for polyubiquitylation by recruiting the target protein directly to the proteasome. Direct-to-proteasome degraders (DPDs) may exhibit different characteristics than ubiquitin-dependent degraders, but few studies of this type of TPD have been published, largely due to the dearth of suitable proteasome ligands. To facilitate studies of DPDs, we report here a mammalian cell line in which the HaloTag protein is fused to the proteasome via Rpn13, one of the ubiquitin receptors. In these cells, a chloroalkane serves as a covalent proteasome ligand surrogate. We show that chimeric molecules comprised of a chloroalkane linked to a ligand for the BET family of proteins or the Cdk2/7/9 family of kinases result in ubiquitin-independent degradation of some of these target proteins. We use this system, the first that allows facile degradation of native proteins in a ubiquitin-independent fashion, to probe two issues: the effect of varying the length of the linker connecting the chloroalkane and the target ligand and the selectivity of degradation within the protein families engaged by the target ligand.

Engineered Protein Fibers with Reinforced Mechanical Properties Via β-Sheet High-Order Assembly.

Protein fibers are ideal alternatives to synthetic polymers due to their unique mechanical properties, biocompatibility, and sustainability. However, engineering biomimetic protein fibers with high mechanical properties remains challenging, particularly in mimicking the high molecular weight of natural proteins and regulating their complex hierarchical structures. Here, a modular design and multi-scale assembly strategy is developed to manufacture robust protein fibers using low- or medium-molecular-weight proteins. The distinct functional and structural properties of flexible, rigid, and cross-linked domains in modular proteins are skillfully harnessed. By regulating the ratio of rigid to flexible domains, the formation of high-order β-sheet crystals aligned along the fiber axis is promoted, enhancing both strength and toughness. Furthermore, the dynamic imine cross-linking network, formed by the aldehyde-amine condensation reaction of the cross-linked domains, further reinforces the protein fibers. Remarkably, fibers spun from modular proteins significantly smaller than natural spidroin exhibit outstanding mechanical properties, surpassing those of protein fibers with same or even higher molecular weights. This strategy offers a promising pathway for fabricating protein fibers suitable for diverse applications.

The benefits of a prolonged-release protein substitute with similarities to natural proteins as a treatment for phenylketonuria: a plain language summary of a study in healthy adults

The benefits of a prolonged-release protein substitute with similarities to natural proteins as a treatment for phenylketonuria: a plain language summary of a study in healthy adults

Bottom-up construction of chiral metal-peptide assemblies from metal cluster motifs.

The exploration of artificial metal-peptide assemblies (MPAs) is one of the most exciting fields because of their great potential for simulating the dynamics and functionality of natural proteins. However, unfavorable enthalpy changes make forming discrete complexes with large and adaptable cavities from flexible peptide ligands challenging. Here, we present a strategy integrating metal-cluster building blocks and peptides to create chiral metal-peptide assemblies and get a family of enantiopure [R-/S-Ni3L2]n (n = 2, 3, 6) MPAs, including the R-/S-Ni6L4 capsule, the S-Ni9L6 trigonal prism, and the R-/S-Ni18L12 octahedron cage. X-ray crystallography shows MPA formation reactions are highly solvent-condition-dependent, resulting in significant changes in ligand conformation and discrete cavity sizes. Moreover, we demonstrate that a structure transformation from Ni18L12 to Ni9L6 in the presence of benzopyrone molecules depends on the peptide conformational selection in crystallization. This work reveals that a metal-cluster building block approach enables facile bottom-up construction of artificial metal-peptide assemblies.

Growth Factor Gene Therapy for Alzheimer's Disease.

Nervous system growth factors are natural proteins of the brain that influence neuronal survival and function throughout life, from embryonic development to old age. In animal models of Alzheimer's disease (AD), the growth factor brain derived neurotrophic factor (BDNF) prevents neuronal death, activates neuronal function, builds new synapses and improves learning and memory. Accordingly, we are determining whether gene delivery of BDNF in patients with AD will slow disease progression and improve memory. In a previous clinical trial of nerve growth factor (NGF) gene therapy in AD patients (NCT00017940, June 2001), we learned that growth factors can unequivocally elicit classic trophic responses from degenerating neurons in AD. Experience gained from the earlier NGF gene therapy trial is guiding our effort to optimize gene delivery of BDNF in our present clinical program (NCT05040217, June 2021).

Charging of DNA Complexes in Positive-Mode Native Electrospray Ionization Mass Spectrometry.

Native mass spectrometry (nMS) provides insights into the structures and dynamics of biomacromolecules in their native-like states by preserving noncovalent interactions through "soft" electrospray ionization (ESI). For native proteins, the number of charges that are acquired scales with the surface area and mass. Here, we explore the effect of highly negatively charged DNA on the ESI charge of protein complexes and find a reduction of the mass-to-charge ratio as well as a greater variation. The charge state distributions of pure DNA assemblies show a lower mass-to-charge ratio than proteins due to their greater density in the gas phase, whereas the charge of protein-DNA complexes can additionally be influenced by the distribution of the ESI charges, ion pairing events, and collapse of the DNA components. Our findings suggest that structural features of protein-DNA complexes can result in lower charge states than expected for proteins.

Dual-Self-Crosslinking Effect of Alginate-Di-Aldehyde with Natural and Synthetic Co-Polymers as Injectable In Situ-Forming Biodegradable Hydrogel.

Injectable, in situ-forming hydrogels, both biocompatible and biodegradable, have garnered significant attention in tissue engineering due to their potential for creating adaptable scaffolds. The adaptability of these hydrogels, made from natural proteins and polysaccharides, opens up a world of possibilities. In this study, sodium alginate was used to synthesize alginate di-aldehyde (ADA) through periodate oxidation, resulting in a lower molecular weight and reduced viscosity, with different degrees of oxidation (54% and 70%). The dual-crosslinking mechanism produced an injectable in situ hydrogel. Initially, physical crosslinking occurred between ADA and borax via borax complexation, followed by chemical crosslinking with gelatin through a Schiff's base reaction, which takes place between the amino groups of gelatin and the aldehyde groups of ADA, without requiring an external crosslinking agent. The formation of Schiff's base was confirmed by Fourier-transform infrared (FT-IR) spectroscopy. At the same time, the aldehyde groups in ADA were characterized using FT-IR, proton nuclear magnetic resonance (¹H NMR), and gel permeation chromatography (GPC), which determined its molecular weight. Furthermore, borax complexation was validated through boron-11 nuclear magnetic resonance (¹¹B NMR). The hydrogel formulation containing 70% ADA, polyethylene glycol (PEG), and 9% gelatin exhibited a decreased gelation time at physiological temperature, attributed to the increased gelatin content and higher degree of oxidation. Rheological analysis mirrored these findings, showing a correlation with gelation time. The swelling capacity was also enhanced due to the increased oxidation degree of PEG and the system's elevated gelatin content and hydrophilicity. The hydrogel demonstrated an average pore size of 40-60 µm and a compressive strength of 376.80 kPa. The lower molecular weight and varied pH conditions influenced its degradation behavior. Notably, the hydrogel's syringeability was deemed sufficient for practical applications, further enhancing its potential in tissue engineering. Given these properties, the 70% ADA/gelatin/PEG hydrogel is a promising candidate and a potential game-changer for injectable, self-crosslinking applications in tissue engineering. Its potential to revolutionize the field is inspiring and should motivate further exploration.

Insights into the binding recognition and computational design of IL-2 muteins with enhanced predicted binding affinity to the IL-2 receptor α

Interleukin-2 (IL-2) is an immune system regulator that has received approval for cancer treatment. However, high-dose IL-2 therapy has seen restricted use due to its low efficacy and on-target toxicity. To enhance the effectiveness of IL-2 therapy, it is essential to engineer IL-2 molecules to enhance their specificity toward target cell populations. In this study, molecular dynamics (MD) simulations and Rosetta software were utilized to design novel high-affinity IL-2Rα-binding IL-2 muteins. MD simulations were used to identify the target residues of IL-2 for design, and Rosetta software were then employed to predict potential IL-2 muteins with higher binding affinity toward IL-2Rα. Rosetta generated two potential designed IL-2 muteins. The results of the MD validation and MM/GBSA analysis indicated that both designed IL-2 muteins exhibited greater predicted binding affinities toward IL-2Rα than that of the native proteins. RMSF analysis demonstrated that the structural fluctuations of free IL-2 and designed muteins were similar, indicating that the mutations did not alter the intramolecular force responsible for IL-2's stability and folding. These designed IL-2 muteins may have potential benefits for cancer immunotherapy.