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Biomolecules Mar 2022Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as...
Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [FeS]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.
Topics: Apoferritins; Cryoelectron Microscopy; Crystallography, X-Ray; Metalloproteins; Oxygen
PubMed: 35327633
DOI: 10.3390/biom12030441 -
Current Opinion in Structural Biology Dec 2021An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method... (Review)
Review
An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.
Topics: Catalysis; Crystallography, X-Ray; Metalloproteins; Spectrum Analysis; Synchrotrons
PubMed: 34455163
DOI: 10.1016/j.sbi.2021.07.007 -
Current Topics in Medicinal Chemistry 2016Metalloproteins have attracted momentous attentions for the treatment of many human diseases, including cancer, HIV, hypertension, etc. This article reviews the... (Review)
Review
Metalloproteins have attracted momentous attentions for the treatment of many human diseases, including cancer, HIV, hypertension, etc. This article reviews the progresses that have been made in the field of drug development of metalloprotein inhibitors, putting emphasis on the targets of carbonic anhydrase, histone deacetylase, angiotensin converting enzyme, and HIV-1 integrase. Many other important metalloproteins are also briefly discussed. The binding and coordination modes of different marketed metalloprotein inhibitors are stated, providing insights to design novel metal binding groups and further novel inhibitors for metalloproteins.
Topics: HIV Infections; Humans; Hypertension; Metalloproteins; Models, Molecular; Molecular Structure; Neoplasms; Protease Inhibitors; Structure-Activity Relationship
PubMed: 26268345
DOI: 10.2174/1568026615666150813145218 -
Inorganic Chemistry Dec 2004
Topics: Chemistry, Inorganic; Metalloproteins; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Folding
PubMed: 15578822
DOI: 10.1021/ic040121q -
Journal of the American Chemical Society Nov 2022Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural...
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)(X) cofactor (dmgH = dimethylglyoxime and X = N, HC, and Pr). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)(CH) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
Topics: Metalloproteins; Crystallography, X-Ray; Escherichia coli; Circular Dichroism; Kinetics
PubMed: 36378237
DOI: 10.1021/jacs.2c08885 -
Current Opinion in Structural Biology Oct 2015Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new... (Review)
Review
Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the sample is destroyed. This review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.
Topics: Binding Sites; Crystallography, X-Ray; Lasers; Metalloproteins; Metals; Models, Molecular; Protein Binding; Protein Conformation; Spectrum Analysis; Temperature
PubMed: 26342144
DOI: 10.1016/j.sbi.2015.07.014 -
Accounts of Chemical Research Apr 2016Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome...
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV-vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.
Topics: Crystallography, X-Ray; Metalloproteins; Protein Conformation; X-Ray Absorption Spectroscopy
PubMed: 26975689
DOI: 10.1021/acs.accounts.5b00538 -
Journal of the American Chemical Society Dec 2022Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins...
Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.
Topics: Metalloproteins; Ligands; Phosphines; Palladium
PubMed: 36417425
DOI: 10.1021/jacs.2c09683 -
Current Opinion in Structural Biology Aug 2001Metalloprotein and redox protein design are rapidly advancing toward the chemical synthesis of novel proteins that have predictable structures and functions. Current... (Review)
Review
Metalloprotein and redox protein design are rapidly advancing toward the chemical synthesis of novel proteins that have predictable structures and functions. Current data demonstrate a breadth of successful approaches to metallopeptide and metalloprotein design based on de novo, rational and combinatorial strategies. These sophisticated synthetic analogs of natural proteins constructively test our comprehension of metalloprotein structure/function relationships. Additionally, designed redox proteins provide novel constructs for examining the thermodynamics and kinetics of biological electron transfer.
Topics: Algorithms; Cysteine; Hemeproteins; Ligands; Metalloproteins; Models, Molecular; Oxidation-Reduction; Protein Engineering; Rod Opsins; Superoxide Dismutase
PubMed: 11495743
DOI: 10.1016/s0959-440x(00)00237-2 -
Nature Chemical Biology Feb 2019Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic...
Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe-2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.
Topics: Amino Acid Sequence; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Escherichia coli; Ferredoxins; Metalloproteins; Mutagenesis, Site-Directed; Protein Processing, Post-Translational
PubMed: 30559426
DOI: 10.1038/s41589-018-0192-3