Naked Metal Cations Swimming in Protein Crystals (original) (raw)
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Metalloprotein Crystallography: More than a Structure
Accounts of Chemical Research, 2016
CONSPECTUS: 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.
Characterization of interactions and metal ion binding sites in proteins
Current Opinion in Structural Biology, 1994
Recent investigations show that as a class of interactions for designing proteins, hydrophobic interactions are not specific enough, hydrophilic interactions are typically too weak, and water interactions are always on the exterior. In terms of overall protein stability, there is a substantial advantage to a nucleus with strong, directional interactions. Metal ion sites in proteins exhibit strong directional preferences for their coordinate ligands, and the specificities manifested by ions have been demonstrated to be useful in reducing molecular fluctuations. The engineered introduction of zinc binding sites has been shown to improve the stabilities of designed proteins. Metal binding sites can therefore provide important structural building blocks for protein design.
Journal of Computational and Theoretical Nanoscience, 2014
HF/3-21g * * was used to study the possible interaction of Ca, Cd and Za with protein. Results indicate that each metal is attached with two hydrogen bondings in two hydrated protein chains. Protein structure has been affected as a result of interaction with the studied metals. The change was noticed in the bond lengths and bond angle of the COOH group. The interaction decreases the calculated band gap energy and increases the total dipole moment which is a good indication for the reactivity of protein after interaction with the studied metals. FTIR verifies experimentally the interaction and indicates that the characteristic bands of metal carboxylate are shifted 190∼200 cm −1 through the lower wavenumbers.
Data mining of metal ion environments present in protein structures
Journal of Inorganic Biochemistry, 2008
Analysis of metal-protein interaction distances, coordination numbers, B-factors (displacement parameters), and occupancies of metal binding sites in protein structures determined by X-ray crystallography and deposited in the PDB shows many unusual values and unexpected correlations. By measuring the frequency of each amino acid in metal ion binding sites, the positive or negative preferences of each residue for each type of cation were identified. Our approach may be used for fast identification of metal-binding structural motifs that cannot be identified on the basis of sequence similarity alone. The analysis compares data derived separately from high and medium resolution structures from the PDB with those from very high resolution small-molecule structures in the Cambridge Structural Database (CSD). For high resolution protein structures, the distribution of metal-protein or metal-water interaction distances agrees quite well with data from CSD, but the distribution is unrealistically wide for medium (2.0 -2.5 Å) resolution data. Our analysis of cation B-factors versus average B-factors of atoms in the cation environment reveals substantial numbers of structures contain either an incorrect metal ion assignment or an unusual coordination pattern. Correlation between data resolution and completeness of the metal coordination spheres is also found.
Structural approaches to probing metal interaction with proteins
Journal of Inorganic Biochemistry, 2012
In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein.
Metallomics, 2011
The peptide was prepared by solid phase peptide synthesis using the Fmoc methodology (Fmoc = 9-fluorenylmethoxycarbonyl). Rink Amide AM resin (Novabiochem, 200-400mesh, loading: 0.68 mmol/g) was used as a solid support. The amino acid building blocks were applied in 4-fold excess over the capacity of the resin. The amino acid residues were coupled to each other (and to the resin) by applying HBTU (3.8 eq./building block), HOBt (4 eq./building block) and N,N-diisopropylethylamine (7.8 eq./building block) in NMP. The Fmoc-protecting groups were removed by using a solution of 20% piperidine in NMP. The usual coupling reaction time was 1 h. The attachment of each amino acid residues was monitored by Kaiser-test (E. Kaiser,
Valence Screening of Water in Protein Crystals Reveals Potential Na+Binding Sites
Journal of Molecular Biology, 1996
Department of Biochemistry comparable electron density of this monovalent cation and water. Valence and Molecular Biophysics calculations can predict the location of metal ion binding sites in proteins Washington University with high precision. These calculations were used to screen 332,242 School of Medicine Box 8231, St. Louis water molecules in 2742 protein structures reported in the Protein Data MO 63110, USA
Prediction of transition metal-binding sites from apo protein structures
Proteins: Structure, Function, and Bioinformatics, 2007
Currently, about 20 novel protein structures are resolved each week by the structural genomics initiative (SGI), a worldwide effort having as one of its goals the creation of a catalog of all protein folds. Functional information for SGI targets is often limited or nonexistent; thus, there is a growing need for procedures to deduce the information directly from the resolved structure. 1 In such instances, initial clues to biochemical function can be sought from ligands and cofactors that often accompany the protein during crystallization. No cofactor group is more prevalent than metal ions, which play crucial roles in enzyme catalysis, molecular regulation, and structure stability. The problem is that a large fraction of metal-binding proteins are resolved in the Protein Data Bank (PDB 2 ) in a prebound (or ''apo'') state with respect to their metal ion cofactors.
De Novo Design and Structural Characterization of Proteins and Metalloproteins
Annual Review of Biochemistry, 1999
▪ De novo protein design has recently emerged as an attractive approach for studying the structure and function of proteins. This approach critically tests our understanding of the principles of protein folding; only in de novo design must one truly confront the issue of how to specify a protein's fold and function. If we truly understand proteins, it should be possible to design receptors, enzymes, and ion channels from scratch. Further, as this understanding evolves and is further refined, it should be possible to design proteins and biomimetic polymers with properties unprecedented in nature.