Protein Folding Research Articles

The role of microRNAs regulation of endoplasmic reticulum stress in ischemia-reperfusion injury: A review.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, responsible for a range of biological functions such as the secretion, modification and folding of proteins, maintaining Ca2+ homeostasis and the synthesis of steroids/lipids, secreted proteins and membrane proteins. When cells are affected by internal or external factors, including abnormal energy metabolism, disrupted Ca2+ balance, altered glycosylation, drug toxicity, and so on, the unfolded or misfolded proteins accumulate in the ER, leading to the unfolded protein response (UPR) and ER stress. The abnormal ER stress has been reported to be involved in various pathological processes. MicroRNAs (miRNAs) are non-coding RNAs with the length of approximately 19-25 nucleotides. They control the expression of multiple genes through posttranscriptional gene silencing in eukaryotes or some viruses. Increasing evidence indicates that miRNAs are involved in various cellular functions and biological processes, such as cell proliferation and differentiation, growth and development, and metabolic homeostasis. Hence, miRNAs participate in multiple pathological processes. Recently, many studies have shown that miRNAs play an important role by regulating ER stress in ischemia-reperfusion (I/R) injury, but the relevant mechanisms are not fully understood. In this review, we reviewed the current understanding of ER stress, as well as the biogenesis and function of miRNAs, and focused on the role of miRNAs regulation of ER stress in I/R injury, with the aim of providing new targets for the treatment of I/R injury.

Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.

Understanding the spleen response of Russian sturgeon (Acipenser gueldenstaedtii) dealing with chronic heat stress and Aeromonas hydrophila challenge

Sturgeon aquaculture has grown in recent years, driven by increasing global demand for its highly valued products. Russian sturgeon (Acipenser gueldenstaedtii), recognised as one of the most valuable species for caviar production, is farmed in several warm-temperate regions. However, the substantial temperature increase due to global warming represents a challenge for developing sturgeon aquaculture. Previously we demonstrated that Russian sturgeon under chronic heat stress (CHS) exhibited a liver metabolic reprogramming to meet energy demands, weakening their innate defences and leading to increased mortality and economic losses. Here, we used RNA-seq technology to analyse regulated genes in the spleen of Russian sturgeons exposed to CHS and challenged with Aeromonas hydrophila. The assembly gave 253,415 unigenes, with 13.7 % having at least one reliable functional annotation. We found that CHS caused mild splenitis and upregulated genes related to protein folding, heat shock response, apoptosis and autophagy while downregulated genes associated with the cell cycle. The cell cycle arrest was maintained upon A. hydrophila challenge in heat-stressed fish, potentially inducing cell senescence. Surprisingly, immunoglobulin heavy and light chains were upregulated in the spleen of stressed sturgeons but not in those maintained at tolerable temperatures; however, no changes in IgM serum levels were observed in any condition. Our findings indicate that long-term exposure to non-tolerable temperatures induced a heat shock response and activated apoptosis and autophagy processes in the spleen. These mechanisms may enable the control of tissue damage and facilitate the recycling of cell components in a condition where the nutrient supply by the liver might be insufficient. Stressed sturgeons challenged with A. hydrophila maintain these mechanisms, which could culminate in cellular senescence.

Engineering Escherichia coli-Derived Nanoparticles for Vaccine Development

The development of effective vaccines necessitates a delicate balance between maximizing immunogenicity and minimizing safety concerns. Subunit vaccines, while generally considered safe, often fail to elicit robust and durable immune responses. Nanotechnology presents a promising approach to address this dilemma, enabling subunit antigens to mimic critical aspects of native pathogens, such as nanoscale dimensions, geometry, and highly repetitive antigen display. Various expression systems, including Escherichia coli (E. coli), yeast, baculovirus/insect cells, and Chinese hamster ovary (CHO) cells, have been explored for the production of nanoparticle vaccines. Among these, E. coli stands out due to its cost-effectiveness, scalability, rapid production cycle, and high yields. However, the E. coli manufacturing platform faces challenges related to its unfavorable redox environment for disulfide bond formation, lack of post-translational modifications, and difficulties in achieving proper protein folding. This review focuses on molecular and protein engineering strategies to enhance protein solubility in E. coli and facilitate the in vitro reassembly of virus-like particles (VLPs). We also discuss approaches for antigen display on nanocarrier surfaces and methods to stabilize these carriers. These bioengineering approaches, in combination with advanced nanocarrier design, hold significant potential for developing highly effective and affordable E. coli-derived nanovaccines, paving the way for improved protection against a wide range of infectious diseases.

Immobilized Horseradish Peroxidase on Enriched Diazo-Activated Silica Gel Harnessed High Biocatalytic Performance at a Steady State in Organic Solvent.

Dimethyldichlorosilane (DMDCS), an efficient silane coupling reagent appearing between the -OH groups of silica gel (SG) and picric acid, instantaneously produces a derivative enriched with nitro groups. The nitro group acting as an end-cap terminates the reaction and subsequently was converted into diazo to couple tyrosine's phenol ring via its O-carbon, the inert center to immobilize horseradish peroxidase (HRP) in a multipoint mode. It maintains the status quo of the native enzyme's protein folding and the entire protein groups' chemistry. The molecular formula of the synthesized material was verified and appeared as {Si(OSi)4 (H2O)x}n{-O-Si(CH3)2-O-C6H2(N+≡N)3(HRP)}4·yH2O; the parameters were evaluated as x = 0.5, n = 1158, and y = 752. The immobilized biocatalyst's activity in organic solvents was 1.5 times better than that in an aqueous medium; it worked smoothly, wherein the activity in both solvents stabilized at six months and continued up to nine months at 63 ± 3% compared to the initial.

Unusual Hydrophobic Property of Blue Fluorescent Amino Acid 4-Cyanotryptophan.

It is a common belief that the negative heat capacity change (ΔCp) associated with protein folding, which is a manifestation of the hydrophobic effect, results from a decrease in the solvent accessible hydrophobic surface area. Herein, we investigate the conformational energy landscape and dynamics of a tetrapeptide composed of two glycine and two 4-cyanotryptophan residues using time-resolved fluorescence spectroscopy, molecular dynamics simulations, and density functional theory calculations and find that, contrary to this expectation, the hydrophobic association of two 4-cyanotryptophan side chains leads to a positive ΔCp (approximately 543 J K-1 mol-1). Furthermore, we find that promoting one of the 4-cyanotryptophans to its excited electronic state strengthens this self-association. Taken together, our results provide not only insight into how modification of an aromatic amino acid can affect its hydrophobicity but also a potential strategy for designing protein sequences that can fold (unfold) at high (low) temperatures.

The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.

Protein disulfide isomerase (PDI) is a member of the mercaptan isomerase family, primarily located in the endoplasmic reticulum (ER). At least 21 PDI family members have been identified. PDI plays a key role in protein folding, correcting misfolded proteins, and catalyzing disulfide bond formation, rearrangement, and breaking. It also acts as a molecular chaperone. Dysregulation of PDI activity is thus linked to diseases such as cancer, infections, immune disorders, thrombosis, neurodegenerative diseases, and metabolic disorders. In particular, elevated intracellular PDI levels can enhance cancer cell proliferation, metastasis, and invasion, making it a potential cancer marker. Cancer cells require extensive protein synthesis, with disulfide bond formation by PDI being a critical producer. Thus, cancer cells have higher PDI levels than normal cells. Targeting PDI can induce ER stress and activate the Unfolded Protein Response (UPR) pathway, leading to cancer cell apoptosis. This review discusses the structure and function of PDI, PDI inhibitors in cancer therapy, and the limitations of current inhibitors, proposing especially future directions for developing new PDI inhibitors.

Site-selectively Phosphorylated Hsp90 C-terminal Domain Variants Provide Access to Deciphering the Chaperone Code.

The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.

Hierarchical Analysis of Protein Structures: From Secondary Structures to Protein Units and Domains.

The three-dimensional structure of proteins is traditionally organized into hierarchical levels, specifically secondary structures and domains. However, different studies suggest the existence of intermediate levels, such as Protein Units (PUs), which provide a refined understanding of protein architecture. PUs, characterized by their compactness and independence, serve as an intermediate organizational level, bridging the gap between secondary structures and domains. This new view not only enhances our comprehension of protein structure, folding, and evolutionary mechanisms but also provides a robust methodology for identifying and categorizing protein domains. Based on the concept of PUs, alternative structural partitioning solutions can be proposed that address the structural ambiguity of proteins, leading to more meaningful domain identification.

Design, synthesis and biological studies of new isoxazole compounds as potent Hsp90 inhibitors.

Heat shock protein 90 (Hsp90), a molecular chaperone, contributes to the preservation of folding, structure, stability, and function proteins. In this study, novel compounds comprising isoxazole structure were designed, synthesized and their potential ability as Hsp90 inhibitors was validated through docking studies. The active site-based compounds were prepared through a multi-step synthesis process and their chemical structures were characterized employing FT-IR, NMR, and mass spectrometry analysis. Cytotoxic and Hsp90 inhibition activities of synthesized compounds were assessed by MTT assay and ELISA kit, respectively. Based on the obtained results, compound 5 exhibited the highest cytotoxicity (IC50; 14 µM) against cancer cells and reduced Hsp90 expression from 5.54 ng/mL in untreated (normal cells) to 1.56 ng/mL in cancer cells. Moreover, molecular dynamics (MD) simulation results indicated its high affinity to target protein and approved its excellent stability which is essential for exerting an inhibitory effect on cancer cell proliferation.

Microgravity and Human Body: Unraveling the Potential Role of Heat-Shock Proteins in Spaceflight and Future Space Missions

In recent years, the increasing number of long-duration space missions has prompted the scientific community to undertake a more comprehensive examination of the impact of microgravity on the human body during spaceflight. This review aims to assess the current knowledge regarding the consequences of exposure to an extreme environment, like microgravity, on the human body, focusing on the role of heat-shock proteins (HSPs). Previous studies have demonstrated that long-term exposure to microgravity during spaceflight can cause various changes in the human body, such as muscle atrophy, changes in muscle fiber composition, cardiovascular function, bone density, and even immune system functions. It has been postulated that heat-shock proteins (HSPs) may play a role in mitigating the harmful effects of microgravity-induced stress. According to past studies, heat-shock proteins (HSPs) are upregulated under simulated microgravity conditions. This upregulation assists in the maintenance of the proper folding and function of other proteins during stressful conditions, thereby safeguarding the physiological systems of organisms from the detrimental effects of microgravity. HSPs could also be used as biomarkers to assess the level of cellular stress in tissues and cells exposed to microgravity. Therefore, modulation of HSPs by drugs and genetic or environmental techniques could prove to be a potential therapeutic strategy to reduce the negative physiological consequences of long-duration spaceflight in astronauts.

Abstract 4136692: Spatial Transcriptomics Identifies Novel Clinical Markers to Classify the Etiology of Rare and Aggressive Cardiac Sarcomas

Background: Cardiac sarcomas, such as leiomyosarcoma (LMS), synovial sarcoma (SS), and undifferentiated pleomorphic sarcoma (UPS), lack sufficient diagnostic markers for early diagnosis and tailored therapeutic interventions. Understanding their molecular signatures with spatial-temporal resolution compared to normal heart tissue is crucial. Hypothesis: Spatial transcriptomics will reveal a comprehensive set of novel clinical markers, including changes in cellular composition, stemness, and metabolic profiles unique for each cardiac sarcoma subtypes. Aims: Identify novel molecular markers for the accurate diagnosis of cardiac sarcoma subtypes. Methods: Using 10X Visium spatial transcriptomics, we analyzed three cardiac sarcoma subtypes (LMS, SS, UPS) and compared them with normal heart tissue. Differential expression analysis identified novel target genes suitable for clinical disease stratification. Subsequently, machine learning models reveal an unbiased transcriptomic profile to distinguish normal from sarcoma tissues. Finally, immunohistochemistry analysis validated the differential marker gene expression. Results: We identify several novel clinical markers, such as IGKC, PCOLCE, NET1, TLE2, MDFI, BGN, TNNC1, and CNN3, that classify normal heart tissue from sarcoma subtypes. Machine learning models and immunostaining analysis confirm the transcriptomic signature in each sarcoma subtype. Pathway enrichment analysis show LMS enriched in antigen processing and B cell-mediated immunity, SS in protein folding and response to incorrect proteins, and UPS in axon development and Wnt signaling regulation. Trajectory Reconstruction Analysis indicate higher stemness scores in all sarcomas, with SS highest, followed by UPS and LMS. Additional cell cycling analysis show LMS cells in G2M phase, SS in G1, and UPS in S phase. SS express a high number of cancer stem markers (EZH2, BMI1, KDM5B, LGR5, HIF1A), while UPS express KDM5B and CTNNB1, and LMS show fewer stem-related marker genes (EZH2, BMI1, KDM5B, CTNNB1). Both SS and LMS express CD44. Glucose and oxidative pathways are also differentially regulated in each sarcoma subtype. Conclusion: Spatial transcriptomics uncovers distinct clinical markers, stemness characteristics, and metabolic activity profiles in cardiac sarcomas, offering insights for fast, accurate and improved diagnostics and therapeutics.

Abstract 4136861: Title: Protein interaction profiling of the Kv11.1 potassium channel reveals new therapeutic targets for Long QT Syndrome

Background: The voltage-gated potassium ion channel K V 11.1 plays a crucial role in cardiac repolarization. Genetic variants in K V 11.1 lead to Long QT Syndrome (LQTS), a condition associated with fatal arrhythmias. Approximately 90% of missense variants linked to LQTS cause intracellular protein transport (trafficking) dysfunction. Some K V 11.1 channel blockers act as chemical chaperones (e.g., E-4031) and increase K V 11.1 channel trafficking. However, these drugs cannot be used in LQTS patients. The underlying mechanisms, such as the protein networks involved in folding and quality control for K V 11.1 channel trafficking remain largely unexplored. Methods: We used affinity-purification (e.g., coimmunoprecipitation) coupled with mass spectrometry to quantify protein interaction changes in human embryonic kidney cells expressing wild-type K V 11.1 or two trafficking-deficient channel variants in the presence or absence of chemical chaperone E-4031. After co-immunoprecipitations and protein digestions, peptides were identified using multiplexed mass spectrometry. Results: We identified 572 protein interactions enriched in K V 11.1-expressing cells. We used bioinformatic analysis to make a cellular model showcasing likely K V 11.1 protein interactions localization from early biogenesis and transcription to plasma membrane expression (Figure 1). We revealed proteins responsible for protein folding, translation, proteasomal degradation, and others with most protein interactions significantly elevated in the genetic variants compared to wild-type (Figure 2). Upon 24-hour treatment with E-4031, some of these interactions were reduced towards wild-type and indicated potential molecular targets for therapeutic intervention (Figure 3). Conclusions: We report the discovery of novel K V 11.1 protein-protein interactions that could be therapeutically targeted to improve K V 11.1 trafficking and treat Long QT Syndrome.

Unlocking marine microbial treasures: new PBP2a-targeted antibiotics elicited by metals and enhanced by RSM-driven transcriptomics and chemoinformatics.

Elicitation through abiotic stress, including heavy metals, is a new natural product drug discovery technique. In this research, three compounds 1, 2, and 6, were achieved by triggering zinc and nickel on marine Sphingomonas sp. and Streptomyces sp., which were absent in normal culture. Compound 5 was obtained for the first time from marine bacteria. All compounds showed potent antibacterial activity against Staphylococcus aureus and bactericidal effect at 300µm, but 6 was more active. The potent compound 6 production was further enhanced through response surface methodology by optimizing the condition consisting of nickel 1mM ions, 20mg/L sucrose, 30mg/L salt and culture time 14days. Under these conditions, the SM-6 production was enhanced with a yield of 6.3mg/L, which was absent in the normal culture. Further transcriptome analysis of compound 6 unveiled its antibacterial activity on S. aureus by modulating heat shock protein genes, disrupting protein folding and synthesis, and perturbing cellular redox balance, leading to a comprehensive inhibition of normal bacterial growth. In addition, ADMET has shown that all compounds are safe for cardiac and hepatotoxicity. To determine the anti-bacterial mechanism, all compounds were docked with PBP2a and DNA gyrase enzyme, and TLR-4 protein for predicting vaccine construct, and the best docking score was achieved against PBP2a enzyme with the highest score of -10.2 for compound 6. In-silico cloning was carried out to ensure the expression of proteins generated and were cloned using S.aureus as a host. The simulation studies have shown that both SM-6-PBP2a and TLR-4-PBP2a complex are stable with the system. This study presents a new approach to anti-bacterial drug discovery from microorganisms through heavy metals triggering and enhancing the compound production through response surface methodology.

A statistical mechanics investigation of unfolded protein response across organisms.

Living systems rely on coordinated molecular interactions, especially those related to gene expression and protein activity. The Unfolded Protein Response is a crucial mechanism in eukaryotic cells, activated when unfolded proteins exceed a critical threshold. It maintains cell homeostasis by enhancing protein folding, initiating quality control, and activating degradation pathways when damage is irreversible. This response functions as a dynamic signaling network, with proteins as nodes and their interactions as edges. We analyze these protein-protein networks across different organisms to understand their intricate intra-cellular interactions and behaviors. In this work, analyzing twelve organisms, we assess how fundamental measures in network theory can individuate seed proteins and specific pathways across organisms. We employ network robustness to evaluate and compare the strength of the investigated protein-protein interaction networks, and the structural controllability of complex networks to find and compare the sets of driver nodes necessary to control the overall networks. We find that network measures are related to phylogenetics, and advanced network methods can identify main pathways of significance in the complete Unfolded Protein Response mechanism.

Protein profile changes during priming explants to embryogenic response in Coffea canephora: identification of the RPN12 proteasome subunit involved in the protein degradation.

Plant somatic embryogenesis encompasses somatic cells switch into embryogenic cells that can later produce somatic embryos with the ability to produce plantlets. Previously, we defined in vitro culture settings for the somatic embryogenesis process of Coffea canephora that comprise adequate plantlets with auxin plus cytokinin followed by cut-leaf explant cultivation with cytokinin, producing embryos with the ability to regenerate plantlets. Here, we confirmed that cultivating cut-leaf explants with cytokinin is sufficient to promote somatic embryos proliferation and the high yield of somatic embryos in the protocol requires adequate plantlets with auxin plus cytokinin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels reveal auxin-plus cytokinin-dependent regulated proteins in plantlets with up and down abundance. Chitinase A class III, proteins involved in the metabolism and folding of proteins, photosynthesis, antioxidant activity, and chromatin organization were identified. The RPN12 protein, which is a subunit of the proteasome 26S, has an abundance that is not associated with transcript changes, suggesting post-translational regulation.

DDDR-41. A GENOME-WIDE CRISPR/CAS9 SCREEN IN GLIOBLASTOMA STEM CELLS UNVEILS SYNTHETIC LETHAL INTERACTIONS AND RESISTANCE MECHANISMS OF JIN001, A BLOOD-BRAIN BARRIER-PENETRANT HSP90 INHIBITOR

Abstract Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive genetic heterogeneity and therapeutic resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes and facilitates the proper folding of numerous proteins, including oncogenic drivers and is overexpressed in cancers. Hsp90 inhibitors can disrupt multiple oncogenic signaling pathways by degrading various client proteins essential for tumor growth and survival, making them attractive therapeutic candidates for GBMs. We have previously reported on the efficacy of Hsp90 inhibitors against gliomas. However, cancer cells develop resistance to Hsp90 inhibitors by upregulating compensatory chaperones (e.g., Hsp70), mutating client proteins, or activating alternative survival pathways, reducing the long-term efficacy of these agents. Combination approaches overcoming such resistance mechanisms are needed. JIN001, a novel Hsp90 inhibitor, demonstrates superior blood-brain barrier (BBB) penetration, and high potential for clinical translation. we performed a genome-wide CRISPR/Cas9 knockout screen using the Toronto Knockout Library v3 (TKOv3) with screen selection pressure from JIN001 in GBM stem cells (GSC272) to identify synthetic lethal interactions and resistance mechanisms associated with JIN001 treatment. Experiments were performed in triplicate with two time points, T22 and T37. Quality control analysis of the NGS data indicated excellent quality and consistency between different replicates. DrugZ analysis revealed a set of sensitive and resistant genes, informing potential combination therapies and further elucidating JIN001’s mechanism of action. Validation studies for the top synthetic lethal targets are underway to confirm the identified interactions and will be presented. This CRISPR-Cas9 screen offers valuable insights into JIN001’s therapeutic potential, reveals new combination strategies to overcome resistance to Hsp90 inhibitors and for use of these agents in other cancers, and paves the way for the development of more effective GBM combination treatment strategies.

Förster Resonance Energy Transfer Measurements in Living Bacteria for Interaction Studies of BamA with BamD and Inhibitor Identification

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamDY184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamDR94, BamDR97, and BamAE127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex.

Differentiation and quantification of bovine and pork gelatin using UPLC-QTOF and ATR-FTIR spectroscopy: Addressing challenges in mixed gelatin analysis and detection

The amino acid sequence dictates protein folding and spatial structure, essential for understanding their behavior. Gelatin, important in industry and biomedicine, demands accurate source authentication due to ethical and dietary concerns. This study explores differentiating and quantifying bovine and pork gelatin using UPLC-QTOF and ATR-FTIR spectroscopy. UPLC-QTOF identified distinct ions for pure bovine (m/z + 962.1471, NRLHFFK) and pork gelatin (m/z + 653.0289, YNEVK) but struggled with mixed gelatins due to protein-protein interactions altering peptide ion profiles. ATR-FTIR was employed to leverage ethanol's differential effects on gelatin's amide bands for quantifying pork gelatin contamination in bovine gelatin. The method showed a strong linear correlation (R2 = 0.9994) with contamination levels and amide band transmission, with detection and quantification limits of 0.85 and 2.85 mg/100 mg, respectively. It effectively identified pork gelatin in halal candy, with recovery rates from 50.05 % to 103.69 %, highlighting ATR-FTIR's potential for accurate gelatin analysis.

Calculating linear and nonlinear multi-ensemble slow collective variables for protein folding.

Traditional molecular dynamics simulation of biomolecules suffers from the conformational sampling problem. It is often difficult to produce enough valid data for post analysis such as free energy calculation and transition path construction. To improve the sampling, one practical solution is putting an adaptive bias potential on some predefined collective variables. The quality of collective variables strongly affects the sampling ability of a molecule in the simulation. In the past, collective variables were built with the sampling data at a constant temperature. This is insufficient because of the same sampling problem. In this work, we apply the standard weighted histogram analysis method to calculate the multi-ensemble averages of pairs of time-lagged features for the construction of both linear and nonlinear slow collective variables. Compared to previous single-ensemble methods, the presented method produces averages with much smaller statistical uncertainties. The generated collective variables help a peptide and a miniprotein fold to their near-native states in a short simulation time period. By using the method, enhanced sampling simulations could be more effective and productive.