17 research outputs found
Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle
Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each Ī±Ī² half of the Ī±2Ī²2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate Ī±Ī² active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves
Sobre as origens e o desenvolvimento do Estado moderno no Ocidente
SĆ£o Paulo - SPRevista Lua NovaNo 1
Characterization of the Zn(II) Binding Properties of the Human Wilmsā Tumor Suppressor Protein C-terminal Zinc Finger Peptide
Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a Ī²Ī²Ī± protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional Ī²Ī²Ī± folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/Ī²Ī²Ī± zinc finger domain, the C-terminal finger of the Wilmsā tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)āWT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)āWT1-4, KfML value of 7.5 Ć 1012 Mā1, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least ā17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)āWT1-4 to those from the model peptide Zn(II)āGGGāCys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ā¤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, ā17.6 kcal/mol
Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-Ī²-lactamase NDM-1
In an effort to characterize the roles of each metal ion in metallo-Ī²-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UVāvis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-Ī²-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data
'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.
The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins
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'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.
The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins
CCDC 866629: Experimental Crystal Structure Determination
Related Article: K.Grubel, A.R.Marts, S.M.Greer, D.L.Tierney, C.J.Allpress, B.J.Laughlin, R.C.Smith, A.M.Arif, L.M.Berreau|2012|Eur.J.Inorg.Chem.||4750|doi:10.1002/ejic.201200212,An entry from the Cambridge Structural Database, the worldās repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures
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Substituent Effects on the Coordination Chemistry of Metal-Binding Pharmacophores
A combination
of XAS, UVāvis, NMR, and EPR was used to examine
the binding of a series of Ī±-hydroxythiones to CoCA. All three
appear to bind preferentially in their neutral, protonated forms.
Two of the three clearly bind in a monodentate fashion, through the
thione sulfur alone. Thiomaltol (TM) appears to show some orientational
preference, on the basis of the NMR, while it appears that thiopyromeconic
acid (TPMA) retains rotational freedom. In contrast, allothiomaltol
(ATM), after initially binding in its neutral form, presumably through
the thione sulfur, forms a final complex that is five-coordinate via
bidentate coordination of ATM. On the basis of optical titrations,
we speculate that this may be due to the lower initial p<i>K</i><sub>a</sub> of ATM (8.3) relative to those of TM (9.0) and TPMA
(9.5). Binding through the thione is shown to reduce the hydroxyl
p<i>K</i><sub>a</sub> by ā¼0.7 pH unit on metal binding,
bringing only ATMās p<i>K</i><sub>a</sub> close to
the pH of the experiment, facilitating deprotonation and subsequent
coordination of the hydroxyl. The data predict the presence of a solvent-exchangeable
proton on TM and TPMA, and Q-band 2-pulse ESEEM experiments on CoCA
+ TM suggest that the proton is present. ESE-detected EPR also showed
a surprising frequency dependence, giving only a subset of the expected
resonances at X-band
āUnconventionalā Coordination Chemistry by Metal Chelating Fragments in a Metalloprotein Active Site
The
binding of three closely related chelators: 5-hydroxy-2-methyl-4<i>H</i>-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4<i>H</i>-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4<i>H</i>-pyran-4-thione (thiopyromeconic acid, TPMA) to the active
site of human carbonic anhydrase II (hCAII) has been investigated.
Two of these ligands display a monodentate mode of coordination to
the active site Zn<sup>2+</sup> ion in hCAII that is not recapitulated
in model complexes of the enzyme active site. This unprecedented binding
mode in the hCAII-thiomaltol complex has been characterized by both
X-ray crystallography and X-ray spectroscopy. In addition, the steric
restrictions of the active site force the ligands into a āflattenedā
mode of coordination compared with inorganic model complexes. This
change in geometry has been shown by density functional computations
to significantly decrease the strength of the metalāligand
binding. Collectively, these data demonstrate that the mode of binding
by small metal-binding groups can be significantly influenced by the
protein active site. Diminishing the strength of the metalāligand
bond results in unconventional modes of metal coordination not found
in typical coordination compounds or even carefully engineered active
site models, and understanding these effects is critical to the rational
design of inhibitors that target clinically relevant metalloproteins
āUnconventionalā Coordination Chemistry by Metal Chelating Fragments in a Metalloprotein Active Site
The
binding of three closely related chelators: 5-hydroxy-2-methyl-4<i>H</i>-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4<i>H</i>-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4<i>H</i>-pyran-4-thione (thiopyromeconic acid, TPMA) to the active
site of human carbonic anhydrase II (hCAII) has been investigated.
Two of these ligands display a monodentate mode of coordination to
the active site Zn<sup>2+</sup> ion in hCAII that is not recapitulated
in model complexes of the enzyme active site. This unprecedented binding
mode in the hCAII-thiomaltol complex has been characterized by both
X-ray crystallography and X-ray spectroscopy. In addition, the steric
restrictions of the active site force the ligands into a āflattenedā
mode of coordination compared with inorganic model complexes. This
change in geometry has been shown by density functional computations
to significantly decrease the strength of the metalāligand
binding. Collectively, these data demonstrate that the mode of binding
by small metal-binding groups can be significantly influenced by the
protein active site. Diminishing the strength of the metalāligand
bond results in unconventional modes of metal coordination not found
in typical coordination compounds or even carefully engineered active
site models, and understanding these effects is critical to the rational
design of inhibitors that target clinically relevant metalloproteins