121 research outputs found
Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters specificity of the reaction pathway
This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Inorganic Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ic301175f.The unique metal abstracting peptide (MAP) asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to DLD-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent due to bis-amide, rather than amine/amide, coordination. In addition, both the Ni-tripeptides and Ni-pentapeptides have a −2 charge. The study here demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in reduction potential for the Ni-pentapeptide. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center
Computational Treatment of Metalloproteins
Metalloproteins present a considerable challenge for modeling, especially when the starting point is far from thermodynamic equilibrium. Examples include formidable problems such as metalloprotein folding and structure prediction upon metal addition, removal, or even just replacement; metalloenzyme design, where stabilization of a transition state of the catalyzed reaction in the specific binding pocket around the metal needs to be achieved; docking to metal-containing sites and design of metalloenzyme inhibitors. Even more conservative computations, such as elucidations of the mechanisms and energetics of the reaction catalyzed by natural metalloenzymes, are often nontrivial. The reason is the vast span of time and length scales over which these proteins operate, and thus the resultant difficulties in estimating their energies and free energies. It is required to perform extensive sampling, properly treat the electronic structure of the bound metal or metals, and seamlessly merge the required techniques to assess energies and entropies, or their changes, for the entire system. Additionally, the machinery needs to be computationally affordable. Although a great advancement has been made over the years, including some of the seminal works resulting in the 2013 Nobel Prize in chemistry, many aforementioned exciting applications remain far from reach. We review the methodology on the forefront of the field, including several promising methods developed in our lab that bring us closer to the desired modern goals. We further highlight their performance by a few examples of applications
The Case of the Missing Ring: Radical Cleavage of a Carbon−Carbon Bond and Implications for GFP Chromophore Biosynthesis
Mechanistic Insights of IscU Conformation Regulation for Fe–S Cluster Biogenesis Revealed by Variable-Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry
ABSTRACT: Iron-sulfur (Fe-S) cluster cofactors are required for the function of many critical cellular processes. These cofactors are assembled and inserted into apo target proteins by conserved Fe-S cluster biosynthetic pathways. In the ISC system of E. coli, the scaffold protein IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. However, there is controversy over whether the structured or disordered form of IscU binds to IscS and functions in Fe-S cluster assembly. The recent development of variable-temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) enables probing the temperature dependence of macromolecular conformation and binding events in a single experiment. Here, vT-ESI nIM-MS results established that IscU exists in structured, intermediate, and disordered conformations. IscU samples shift towards high-charge states that have more extended conformations under extreme temperatures, consistent with cold/heat denaturation. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding (i) attenuates the cold/heat-denaturation of IscU and promotes the refolding of IscU; (ii) favors the structured aand intermediate conformations and inhibits the disordered high charge states; and (iii) attenuates collisional induced unfolding (CIU) of intermediate conformations. Moreover, IscS was shown to alter the local conformation around the IscU active site, resulting in weakened Zn(II) affinity. Overall, these findings provide structural rationalization of the role of Zn(II) on stabilizing IscU conformation and the role of IscS on altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nMS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding
Kinetic Isotope Effect Studies on the de Novo Rate of Chromophore Formation in Fast- and Slow-Maturing GFP Variants
Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures
Green fluorescent protein has revolutionized cell labeling and molecular tagging, yet the driving force and mechanism for its spontaneous fluorophore synthesis are not established. Here we discover mutations that substantially slow the rate but not the yield of this posttranslational modification, determine structures of the trapped precyclization intermediate and oxidized postcyclization states, and identify unanticipated features critical to chromophore maturation. The protein architecture contains a dramatic ≈80° bend in the central helix, which focuses distortions at G67 to promote ring formation from amino acids S65, Y66, and G67. Significantly, these distortions eliminate potential helical hydrogen bonds that would otherwise have to be broken at an energetic cost during peptide cyclization and force the G67 nitrogen and S65 carbonyl oxygen atoms within van der Waals contact in preparation for covalent bond formation. Further, we determine that under aerobic, but not anaerobic, conditions the Gly-Gly-Gly chromophore sequence cyclizes and incorporates an oxygen atom. These results lead directly to a conjugation-trapping mechanism, in which a thermodynamically unfavorable cyclization reaction is coupled to an electronic conjugation trapping step, to drive chromophore maturation. Moreover, we propose primarily electrostatic roles for the R96 and E222 side chains in chromophore formation and suggest that the T62 carbonyl oxygen is the base that initiates the dehydration reaction. Our molecular mechanism provides the basis for understanding and eventually controlling chromophore creation
Understanding Blue-to-Red Conversion in Monomeric Fluorescent Timers and Hydrolytic Degradation of Their Chromophores
The mechanism of cyclization in chromophore maturation of green fluorescent protein: A theoretical study
An intriguing aspect of the green fluorescent protein (GFP) is the autocatalytic post-translational modification that results in the formation of its chromophore. Numerous experimental and theoretical studies indicate that cyclization is the first and the most important step in the maturation process. In this work, two proposed mechanisms for the cyclization were investigated by using the hybrid density functional theory method B3LYP. Cluster models corresponding to the two mechanisms proposed by Wachter et al. [J. Biol. Chem. 2005, 280, 26248-26255] are constructed on the basis of the X-ray crystal structure (PDB entry 2AWJ) and corresponding reaction path potential energy profiles for the two cyclization mechanisms are presented. Our results suggest that the backbone condensation initiated by deprotonation of the Gly67 amide nitrogen is easier than deprotonation of the Tyr66 α-carbon. Moreover, Arg96 fulfills the role of stabilizing the enolate moiety, and Glu222 plays the role of a general base. The formation of the cyclized product is found to be 16.0 and 18.6 kcal/mol endothermic with respect to the two models, which is in agreement with experimental observation
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