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Angewandte Chemie (International Ed. in... Feb 2021De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control...
De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O reduction when adsorbed onto a pyrolytic graphite electrode.
Topics: Amino Acid Sequence; Catalysis; Cysteine; Electron Spin Resonance Spectroscopy; Heme; Histidine; Hydrogen-Ion Concentration; Iron; Metalloproteins; Oxidation-Reduction; Oxygen; Peptides; Protein Conformation, alpha-Helical
PubMed: 33215801
DOI: 10.1002/anie.202012673 -
Journal of Chromatography. B,... Mar 1997Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were...
Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3-10 and 5-8. Several different proteins and one peptide with known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3-10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe3+/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3-10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (diferric forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5-8). Rabbit liver MT subjected to cIEF with a pH gradient of 3-10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3-10, heterogeneous peaks with higher pI values (4.12-4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using a pH gradient of 5-8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.
Topics: Animals; Conalbumin; Electrophoresis, Capillary; Horses; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Metallothionein; Rabbits; Transferrin
PubMed: 9106028
DOI: 10.1016/s0378-4347(96)00407-0 -
Chemical Reviews Apr 2014
Review
Topics: Electron Transport; Electrons; Kinetics; Metalloproteins; Models, Molecular; Protein Structure, Tertiary; Thermodynamics
PubMed: 24279515
DOI: 10.1021/cr4004715 -
Methods in Enzymology 1993
Review
Topics: Animals; Cold Temperature; Metalloproteins; Molecular Structure; Myoglobin; Spectrophotometry; Thermodynamics; Vitamin B 12
PubMed: 8277887
DOI: 10.1016/0076-6879(93)26007-v -
Biosensors & Bioelectronics Jan 2022The development of cell-based bioelectronic devices largely depends on the direct control of intracellular redox states. However, most related studies have focused on...
Bionanohybrid composed of metalloprotein/DNA/MoS/peptides to control the intracellular redox states of living cells and its applicability as a cell-based biomemory device.
The development of cell-based bioelectronic devices largely depends on the direct control of intracellular redox states. However, most related studies have focused on the accurate measurement of electrical signals from living cells, whereas direct intracellular state control remains largely unexplored. Here, we developed a biocompatible transmembranal bionanohybrid structure composed of a recombinant metalloprotein, DNA, molybdenum disulfide nanoparticles (MoS), and peptides to control intracellular redox states, which can be used as a cell-based biomemory device. Using the capacitance of MoS located inside the cell, the bionanohybrid controled the intracellular redox states of living cells by recording and extracting intracellular charges, which inturn was achieved by activating (writing) and deactivating (erasing) the cells. As a proof of concept, cell-based biomemory functions including writing, reading, and erasing were successfully demonstrated and confirmed via electrochemical methods and patch-clamp analyses, resulting in the development of the first in vitro cell-based biomemory device. This newly developed bionanohybrid provides a novel approach to control cellular redox states for cell-based bioelectronic applications, and can be applicable in a wide range of biological fields including bioelectronic medicine and intracellular redox status regulation.
Topics: Biosensing Techniques; DNA; Metalloproteins; Molybdenum; Oxidation-Reduction; Peptides
PubMed: 34678652
DOI: 10.1016/j.bios.2021.113725 -
Journal of Inorganic Biochemistry Oct 2012Structures of metalloprotein active sites derived from X-ray crystallography frequently contain chemical anomalies such as unexpected atomic geometries or elongated...
Structures of metalloprotein active sites derived from X-ray crystallography frequently contain chemical anomalies such as unexpected atomic geometries or elongated bond-lengths. Such anomalies are expected from the known errors inherent in macromolecular crystallography (ca. 0.1-0.2Å) and from the lack of appropriate restraints for metal sites which are often without precedent in the small molecule structure literature. Here we review the potential of X-ray absorption spectroscopy to provide information and perspective which could aid in improving the accuracy of metalloprotein crystal structure solutions. We also review the potential problem areas in analysis of the extended X-ray absorption fine structure (EXAFS) and discuss the use of density functional theory as another possible source of geometrical restraints for crystal structure analysis of metalloprotein active sites.
Topics: Catalytic Domain; Crystallography, X-Ray; Databases, Protein; Metalloproteins; X-Ray Absorption Spectroscopy
PubMed: 22824156
DOI: 10.1016/j.jinorgbio.2012.06.019 -
Advances in Biochemical... 2023Metalloproteins, proteins containing metal atoms or clusters within their structures, are critical for various biological functions across all domains of life. More than...
Metalloproteins, proteins containing metal atoms or clusters within their structures, are critical for various biological functions across all domains of life. More than hundreds of different types have been discovered, which conduct various roles such as transportation of O, catalyzing chemical reactions, sensing environmental changes, and relaying electrons. Metalloprotein molecules incorporate a variety of metal atoms, coordinated to specific amino acid residues that affect their conformation and functionality. The process of metal incorporation typically occurs during or post-protein folding, often requiring chaperones for metal ion delivery and quality control. Progress in understanding metal incorporation and metalloprotein functionality has been enhanced by cell-free protein synthesis (CFPS) methods that offer direct control over the synthesis environment. This chapter reviews the diverse applications of CFPS methods in metalloprotein research, encompassing structure-function studies, protein engineering, and creation of artificial metalloproteins. Examples demonstrating the utility and advances brought about by CFPS in synthetic biology, electrochemistry, and drug discovery are highlighted. Despite remarkable progress, challenges remain in optimizing and advancing the CFPS methods, underscoring the need for future explorations in this transformative approach to metalloprotein study and engineering.
Topics: Metalloproteins; Metals; Amino Acids
PubMed: 37561181
DOI: 10.1007/10_2023_233 -
Current Opinion in Chemical Biology Apr 2015This review presents recent examples of metal-binding promiscuity in protein scaffolds and highlights the effect of metal variation on catalytic functionality. Naturally... (Review)
Review
This review presents recent examples of metal-binding promiscuity in protein scaffolds and highlights the effect of metal variation on catalytic functionality. Naturally evolved binding sites, as well as unnatural amino acids and cofactors can bind a diverse range of metals, including non-biological transition elements. Computational screening and rational design have been successfully used to create promiscuous binding-sites. Incorporation of non-native metals into proteins expands the catalytic range of transformations catalysed by enzymes and enhances their potential for application in chemicals synthesis.
Topics: Amino Acids; Enzymes; Metalloproteins; Metals; Protein Engineering; Substrate Specificity
PubMed: 25603469
DOI: 10.1016/j.cbpa.2014.12.035 -
Journal of Chemical Information and... Mar 2024Metalloproteins play a fundamental role in molecular biology, contributing to various biological processes. However, the discovery of high-affinity ligands targeting...
Metalloproteins play a fundamental role in molecular biology, contributing to various biological processes. However, the discovery of high-affinity ligands targeting metalloproteins has been delayed due, in part, to a lack of suitable tools and data. Molecular docking, a widely used technique for virtual screening of small-molecule ligand interactions with proteins, often faces challenges when applied to metalloproteins due to the particular nature of the ligand metal bond. To address these limitations associated with docking metalloproteins, we introduce a knowledge-driven docking approach known as "metalloprotein bias docking" (MBD), which extends the AutoDock Bias technique. We assembled a comprehensive data set of metalloprotein-ligand complexes from 15 different metalloprotein families, encompassing Ca, Co, Fe, Mg, Mn, and Zn metal ions. Subsequently, we conducted a performance analysis of our MBD method and compared it to the conventional docking (CD) program AutoDock4, applied to various metalloprotein targets within our data set. Our results demonstrate that MBD outperforms CD, significantly enhancing accuracy, selectivity, and precision in ligand pose prediction. Additionally, we observed a positive correlation between our predicted ligand free energies and the corresponding experimental values. These findings underscore the potential of MBD as a valuable tool for the effective exploration of metalloprotein-ligand interactions.
Topics: Humans; Metalloproteins; Molecular Docking Simulation; Ligands
PubMed: 38373276
DOI: 10.1021/acs.jcim.3c01853 -
Metallomics : Integrated Biometal... Jan 2013
Topics: Animals; Humans; Metalloproteins; Metals; Periodicals as Topic; Proteomics
PubMed: 23238130
DOI: 10.1039/c2mt90067j