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Journal of Chemical Information and... May 2024A substantial portion of various organisms' proteomes comprises intrinsically disordered proteins (IDPs) that lack a defined three-dimensional structure. These IDPs...
A substantial portion of various organisms' proteomes comprises intrinsically disordered proteins (IDPs) that lack a defined three-dimensional structure. These IDPs exhibit a diverse array of conformations, displaying remarkable spatiotemporal heterogeneity and exceptional conformational flexibility. Characterizing the structure or structural ensemble of IDPs presents significant conceptual and methodological challenges owing to the absence of a well-defined native structure. While databases such as the Protein Ensemble Database (PED) provide IDP ensembles obtained through a combination of experimental data and molecular modeling, the absence of reaction coordinates poses challenges in comprehensively understanding pertinent aspects of the system. In this study, we leverage the energy landscape visualization method (JCTC, 6482, 2019) to scrutinize four IDP ensembles sourced from PED. ELViM, a methodology that circumvents the need for a priori reaction coordinates, aids in analyzing the ensembles. The specific IDP ensembles investigated are as follows: two fragments of nucleoporin (NUL: 884-993 and NUS: 1313-1390), yeast sic 1 N-terminal (1-90), and the N-terminal SH3 domain of Drk (1-59). Utilizing ELViM enables the comprehensive validation of ensembles, facilitating the detection of potential inconsistencies in the sampling process. Additionally, it allows for identifying and characterizing the most prevalent conformations within an ensemble. Moreover, ELViM facilitates the comparative analysis of ensembles obtained under diverse conditions, thereby providing a powerful tool for investigating the functional mechanisms of IDPs.
Topics: Proteins; Databases, Protein; Models, Molecular; Protein Structure, Tertiary; Saccharomyces cerevisiae; Protein Unfolding
PubMed: 38713459
DOI: 10.1021/acs.jcim.4c00080 -
International Journal of Biological... Jun 2024In this study, we examined the possibility of using industrial microwave processing to enhance the gelling properties and reduce the starch digestibility of mung bean...
Enhancing the nonlinear rheological property and digestibility of mung bean flour gels using controlled microwave treatments: Effect of starch debranching and protein denaturation.
In this study, we examined the possibility of using industrial microwave processing to enhance the gelling properties and reduce the starch digestibility of mung bean flour (MBF). MBF (12.6 % moisture) was microwaved at a power of 6 W/g to different final temperatures (100-130 °C), and then its structural and functional properties were characterized. The microwave treatment had little impact on the crystalline structure or amylose content of the starch, but it roughened the starch granule surfaces and decreased the short-range ordered structure and degree of branching. In addition, the extent of mung bean protein denaturation caused by the microwave treatment depended on the final temperature. Slightly denaturing the proteins (100 °C) did not affect the nature of the gels (protein phase dispersed in a starch phase) but the gel network became more compact. Moderately denaturing the proteins (110-120 °C) led to more compact and homogeneous starch-protein double network gels. Excessive protein denaturation (130 °C) caused the gel structure to become more heterogeneous. As a result, the facilitated tangles between starch chains by more linear starch molecules after debranching, and the protein network produced by moderate protein denaturation led to the formation of stronger gel and the improvement of plasticity during large deformation (large amplitude oscillatory shear-LAOS). Starch recrystallization, lipid complexion, and protein network retard starch digestion in the MBF gels. In conclusion, an industrial microwave treatment improved the gelling and digestive properties of MBF, and Lissajous curve has good adaptability in characterizing the viscoelasticity of gels under large deformations.
Topics: Microwaves; Starch; Rheology; Vigna; Gels; Flour; Protein Denaturation; Plant Proteins; Temperature; Amylose
PubMed: 38704060
DOI: 10.1016/j.ijbiomac.2024.132049 -
Journal of Molecular Biology Jun 2024Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus...
Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phosphosites in NPM1 reside within signal sequences, this work suggests a mechanism of NPM1 regulation by which NPM1 phosphorylation can promote 14-3-3 binding to affect NPM1 shuttling between cell compartments. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.
Topics: Humans; 14-3-3 Proteins; Binding Sites; Nucleophosmin; Phosphorylation; Protein Binding; Protein Multimerization
PubMed: 38702038
DOI: 10.1016/j.jmb.2024.168592 -
Science Advances May 2024The AAA-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to...
The AAA-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.
Topics: Valosin Containing Protein; Humans; Cytosol; Cell Nucleus; Mutation; Active Transport, Cell Nucleus; DNA Damage; Adenosine Triphosphatases; Cell Cycle Proteins; Protein Transport; Nuclear Proteins; DNA Repair; Autophagy; Protein Binding; HEK293 Cells; Adaptor Proteins, Signal Transducing
PubMed: 38701207
DOI: 10.1126/sciadv.adl6082 -
The Journal of Physical Chemistry... May 2024Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent...
Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent interactions, and thus native fold features, are believed to be largely retained upon desolvation. However, the conformation usually depends heavily on the charge state of the species investigated. In this study, we combine transition metal ion Förster resonance energy transfer (tmFRET) and ion mobility-mass spectrometry (IM-MS) with molecular dynamics (MD) simulations to interrogate the β-hairpin structure of GB1p in vacuo. Fluorescence lifetime values and collisional cross sections suggest an unfolding of the β-hairpin motif for higher charge states. MD simulations are consistent with experimental constraints, yet intriguingly provide an alternative structural interpretation: preservation of the β-hairpin is not only predicted for 2+ but also for 4+ charged species, which is unexpected given the substantial Coulomb repulsion for small secondary structure scaffolds.
Topics: Molecular Dynamics Simulation; Fluorescence Resonance Energy Transfer; Protein Structure, Secondary; Mass Spectrometry
PubMed: 38700091
DOI: 10.1021/acs.jpclett.4c00920 -
International Journal of Biological... May 2024In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked...
In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked at 55 °C (B-55) and 85 °C (B-85) showed an increase of β-sheet content by 3.2 % and a decrease of α-helix content by 3.3 %, indicating that proteins were gradually unfolded while oleosomes remained intact. The protein resisted thermal denaturation during secondary heating, and soymilks were stable as reflected by a small d (0.4 μm). However, raw soymilk from soybeans baked at 115 °C (B-115), steamed for 1 min (ST-1) and 5 min (ST-5) presented oleosomes destruction and lipids aggregates. The proteins were coated around the oil aggregates. The β-turn content from soybeans steamed for 10 min (ST-10) increased by 9.5 %, with a dense network where the OBs were tightly wrapped, indicating the serious protein denaturation. As a result, the soymilks B-115 or steamed ones were unstable as evidenced by the serious protein aggregation and larger d (5.65-12.48 μm). Furthermore, the soymilks were graininess and the protein digestion was delayed due to the formation of insoluble protein aggregates. The flavor and early-stage lipid digestion of soymilk from steamed soybeans was improved owing to lipid release.
Topics: Protein Denaturation; Soy Milk; Soybean Proteins; Hot Temperature; Lipid Droplets; Cooking
PubMed: 38697416
DOI: 10.1016/j.ijbiomac.2024.131999 -
Journal of Oleo Science 2024Protein soils must be removed for both appearance and hygienic reasons. They are denatured by heat treatment or bleaching and cleaned using enzymes. Among the various...
Protein soils must be removed for both appearance and hygienic reasons. They are denatured by heat treatment or bleaching and cleaned using enzymes. Among the various types of protein soils, blood soils are the most noticeable and known to be denatured by heat and bleaching by oxidation. We verified herein that the detergency of heat and oxidatively denatured hemoglobin is greatly improved by the enzyme immersing treatment in the detergency with SDS and can be analyzed using the probability density functional method. The probability density functional method evaluates the cleaning power by assuming that the adhesion and cleaning force of soils are not uniquely determined, but instead have a distribution in intensity, with a usefulness that had recently been demonstrated. This analytical method showed that the cleaning power of the enzyme immersing treatment improved when the soil adhesive force was decreased due to denatured protein degradation, even though the cleaning power of the SDS remained unchanged, and the values were consistent with those in the cleaning test. In conclusion, the probability density functional method can be used to analyze enzymatic degradation of denatured protein soils and the resulting changes in their detergency.
Topics: Protein Denaturation; Sodium Dodecyl Sulfate; Oxidation-Reduction; Hot Temperature; Hemoglobins; Soil; Probability
PubMed: 38692891
DOI: 10.5650/jos.ess23262 -
Proceedings of the National Academy of... May 2024Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these...
Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.
Topics: Nanoparticles; Molecular Chaperones; Polymers; Protein Denaturation; Protein Refolding; Protein Folding; Cytochromes c; Laccase; Lipase
PubMed: 38691587
DOI: 10.1073/pnas.2403049121 -
Journal of Biomolecular Structure &... Apr 2024In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy....
In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein's natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.Communicated by Ramaswamy H. Sarma.
PubMed: 38682862
DOI: 10.1080/07391102.2024.2335304 -
ACS Central Science Apr 2024The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the...
The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the translocation of a protein involves two discrete steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules can achieve high protein uptake, but few are capable of efficient endosomal escape. One exception to this rule is ZF5.3, a mini-protein that exploits elements of the natural endosomal maturation machinery to translocate across endosomal membranes. Although some ZF5.3-protein conjugates are delivered efficiently to the cytosol or nucleus, overall delivery efficiency varies widely for different cargoes with no obvious design rules. Here we show that delivery efficiency depends on the ability of the cargo to unfold. Using fluorescence correlation spectroscopy, a single-molecule technique that precisely measures intracytosolic protein concentration, we show that regardless of size and pI, low- cargoes of ZF5.3 (including intrinsically disordered domains) bias endosomal escape toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal, even if the is high. These findings imply a novel pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.
PubMed: 38680556
DOI: 10.1021/acscentsci.4c00016