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Free Radical Biology & Medicine Aug 2020Large-size subunit catalases (LSCs) have a C-terminal domain that is structurally similar to DJ-1 and Hsp31 proteins, which have well documented molecular chaperone...
Large-size subunit catalases (LSCs) have a C-terminal domain that is structurally similar to DJ-1 and Hsp31 proteins, which have well documented molecular chaperone activity. Like chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in different microorganisms. Here we document that the C-terminal domain of LSCs assist other proteins to preserve their active conformation. Heat, urea, or HO denaturation of alcohol dehydrogenase was prevented by LSCs or the C-terminal domain of Catalase-3 (TDC3); in contrast, small-size subunit catalases (SSCs) or LSCs without the C-terminal domain (C3 or C63) did not have this effect. Similar results were obtained if the alcohol dehydrogenase was previously denatured by heat and then the different catalases or truncated enzymes were added. The TDC3 also protected both the C3 and the bovine liver catalase from heat denaturation. The chaperone activity of CAT-3 or the TDC3 increased survival of E. coli under different stress conditions whereas the C3 did not. It is concluded that the C-terminal domain of LSCs has a chaperone activity that is instrumental for cellular resistance to stress conditions, such as oxidative stress that leads to cell differentiation in filamentous fungi.
Topics: Animals; Catalase; Cattle; Escherichia coli; Hydrogen Peroxide; Molecular Chaperones; Protein Folding
PubMed: 32502516
DOI: 10.1016/j.freeradbiomed.2020.05.020 -
Nature Communications Feb 2023DNA origami may enable more versatile gene delivery applications through its ability to create custom nanoscale objects with specific targeting, cell-invading, and...
DNA origami may enable more versatile gene delivery applications through its ability to create custom nanoscale objects with specific targeting, cell-invading, and intracellular effector functionalities. Toward this goal here we describe the expression of genes folded in DNA origami objects delivered to mammalian cells. Genes readily express from custom-sequence single-strand scaffolds folded within DNA origami objects, provided that the objects can denature in the cell. We demonstrate enhanced gene expression efficiency by including and tuning multiple functional sequences and structures, including virus-inspired inverted-terminal repeat-like (ITR) hairpin motifs upstream or flanking the expression cassette. We describe gene-encoding DNA origami bricks that assemble into multimeric objects to enable stoichiometrically controlled co-delivery and expression of multiple genes in the same cells. Our work provides a framework for exploiting DNA origami for gene delivery applications.
Topics: Nanotechnology; Nucleic Acid Conformation; DNA; Nanostructures
PubMed: 36823187
DOI: 10.1038/s41467-023-36601-1 -
Chem & Bio Engineering Mar 2024Enzymes, as highly efficient biocatalysts, excel in catalyzing diverse reactions with exceptional activity and selective properties under mild conditions. Nonetheless,... (Review)
Review
Enzymes, as highly efficient biocatalysts, excel in catalyzing diverse reactions with exceptional activity and selective properties under mild conditions. Nonetheless, their broad applications are hindered by their inherent fragility, including low thermal stability, limited pH tolerance, and sensitivity to organic solvents and denaturants. Encapsulating enzymes within metal-organic frameworks (MOFs) can protect them from denaturation in these harsh environments. However, this often leads to a compromised enzyme activity. In recent years, extensive research efforts have been dedicated to enhancing enzymatic activity within MOFs, leading to the development of new enzyme-MOF composites that not only preserve their catalytic potential but also outperform their free counterparts. This Review provides a comprehensive review on recent developments in enzyme-MOF composites with a specific emphasis on their enhanced enzymatic activity compared to free enzymes.
PubMed: 38566967
DOI: 10.1021/cbe.3c00091 -
Molecules (Basel, Switzerland) Dec 2022The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an...
The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman's rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.
Topics: In Situ Hybridization, Fluorescence; DNA; Oligonucleotides; Nucleotides; DNA, Complementary; DNA Probes
PubMed: 36615321
DOI: 10.3390/molecules28010127 -
Journal of Inorganic Biochemistry Oct 2020We have analyzed the early stages of unfolding of cytochromes c-b (PDB ID: 2BC5) and Rd apo b (PDB ID: 1YYJ). Our geometrical approach proceeds from an analysis of the...
We have analyzed the early stages of unfolding of cytochromes c-b (PDB ID: 2BC5) and Rd apo b (PDB ID: 1YYJ). Our geometrical approach proceeds from an analysis of the crystal structure reported for each protein. We quantify, residue-by-residue and region-by-region, the spatial and angular changes in the structure as the protein denatures, and quantify differences that result from the seven residues that differ in the two proteins. Using two independent analyses, one based on spatial metrics and the second on angular metrics, we establish the order of unfolding of the five helices in cyt c-b and the four helices in the apo protein. For the two helices nearest the N-terminal end of both proteins, the ones in the apo protein unfold first. For the two helices nearest the C-terminal end, the interior helix of the apo protein unfolds first, whereas the terminal helix of the holo protein unfolds first. Excluded-volume effects (repulsive interactions) are minimized in turning regions; the overall range in Δ values is Δ = 36.3 Å for cyt c-b and Δ = 36.6 Å for the apo protein, whereas the span for all 20 amino acids is Δ = 167.7 Å. As our work indicates that the interior helix of cytochrome c-b is the first to fold, we suggest that this helix protects the heme from misligation, consistent with ultrafast folding over a minimally frustrated funneled landscape.
Topics: Apoproteins; Crystallography, X-Ray; Cytochrome b Group; Cytochromes c; Escherichia coli; Escherichia coli Proteins; Heme; Kinetics; Models, Molecular; Protein Folding
PubMed: 32818710
DOI: 10.1016/j.jinorgbio.2020.111209 -
Nature Communications Jan 2022Engineering shape memory/morphing materials have achieved considerable progress in polymer-based systems with broad potential applications. However, engineering...
Engineering shape memory/morphing materials have achieved considerable progress in polymer-based systems with broad potential applications. However, engineering protein-based shape memory/morphing materials remains challenging and under-explored. Here we report the design of a bilayer protein-based shape memory/morphing hydrogel based on protein folding-unfolding mechanism. We fabricate the protein-bilayer structure using two tandem modular elastomeric proteins (GB1) and (FL). Both protein layers display distinct denaturant-dependent swelling profiles and Young's moduli. Due to such protein unfolding-folding induced changes in swelling, the bilayer hydrogels display highly tunable and reversible bidirectional bending deformation depending upon the denaturant concentration and layer geometry. Based on these programmable and reversible bending behaviors, we further utilize the protein-bilayer structure as hinge to realize one-dimensional to two-dimensional and two-dimensional to three-dimensional folding transformations of patterned hydrogels. The present work will offer new inspirations for the design and fabrication of novel shape morphing materials.
Topics: Amino Acid Sequence; Elastic Modulus; Elastomers; Hydrogels; Polymers; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Folding; Protein Unfolding; Proteins; Wettability
PubMed: 35013234
DOI: 10.1038/s41467-021-27744-0 -
Medical Anthropology Quarterly Dec 2021This article presents ethnographic research with in situ seed savers and seed activists in London. Unpicking the knotty relations between kinship, place, and generation...
This article presents ethnographic research with in situ seed savers and seed activists in London. Unpicking the knotty relations between kinship, place, and generation among seed savers and their seeds, this article focuses on how the intrinsic and extrinsic get woven through generations and how the situ or environment of an entity is (and is not) recognized in its identity and multispecies kin relations. I argue that thinking about seeds and the worlds in which they grow suggests that they are not only embedded in their environments, but also embody their environments. If seeds bring with them their worlds, then they are inherently malleable, so seed savers are concerned about how commercial seed breeding and ex situ conservation denatures seeds' embodied relationships with their environments and, with that, their inherent intergenerational malleability. [seed saving, multispecies kinship, generation, environment].
Topics: Anthropology, Medical; Humans; London; Seeds
PubMed: 35066931
DOI: 10.1111/maq.12684 -
Nucleic Acids Research Jan 2021ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84%...
ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84% from the previous version. It contains several thermodynamic parameters such as melting temperature, free energy obtained with thermal and denaturant denaturation, enthalpy change and heat capacity change along with experimental methods and conditions, sequence, structure and literature information. Besides, the current version of the database includes about 120 000 thermodynamic data obtained for different organisms and cell lines, which are determined by recent high throughput proteomics techniques using whole-cell approaches. In addition, we provided a graphical interface for visualization of mutations at sequence and structure levels. ProThermDB is cross-linked with other relevant databases, PDB, UniProt, PubMed etc. It is freely available at https://web.iitm.ac.in/bioinfo2/prothermdb/index.html without any login requirements. It is implemented in Python, HTML and JavaScript, and supports the latest versions of major browsers, such as Firefox, Chrome and Safari.
Topics: Databases, Protein; Information Storage and Retrieval; Mutant Proteins; Proteins; Statistics as Topic; Thermodynamics
PubMed: 33196841
DOI: 10.1093/nar/gkaa1035 -
Protein Science : a Publication of the... Apr 2023A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher-order structures and... (Review)
Review
A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher-order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the "bottom-up" approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher-order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the "top-down" approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in-depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero-complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near-native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.
Topics: Tandem Mass Spectrometry; Peptides; Proteome; Protein Processing, Post-Translational
PubMed: 36851867
DOI: 10.1002/pro.4612 -
Frontiers in Bioengineering and... 2023Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant... (Review)
Review
Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.
PubMed: 37545893
DOI: 10.3389/fbioe.2023.1249196