The Limitations of X-ray Absorption Spectroscopy for Determining the Structure of Zinc Sites in Proteins. When Is a Tetrathiolate Not a Tetrathiolate?

Clark-Baldwin, Kimber; Tierney, David L.; Govindaswamy, Nandakumar; Gruff, Eric S.; Kim, Chongwoo; Berg, Jeremy M.; Koch, Stephen A.; Penner‐Hahn, James E.

doi:10.1021/ja980580o

Cited by 129 publications

(207 citation statements)

References 52 publications

(69 reference statements)

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“…Assemblies formed from initial Zn 2+ to peptide ratios of 1.0 gave a final ratio between 0.5 and 0.6, slightly lower than that of the Aβ(13-21)K16A peptide. 20 The XAS results for the Zn 2+-fibrillar complex of Ac-Aβ(13-21)H14A were also consistent with a first Zn coordination shell of 3N(Im)/1O or 2N(Im)/2O atoms ( Figure S3 and Table S1). The appearance of the 3 and 4 Å peaks in the Fourier transform of the EXAFS again were diagnostic for two imidazoles, consistent with two His13's on one face of the β-sheet chelating Zn 2+ and similar to that seen for Aβ(13-21)K16A at low [Zn 2+ ].…”

supporting

confidence: 53%

“…The intensity and the position of the absorbance at the Zn K-edge region and EXAFS were consistent with four light elements (nitrogen or oxygen) in the coordination sphere of Zn 2+ in the fibers/ribbons (Figure 4a), as well as the supernatant samples. 20,21 Curve fitting indicated either 3N(Im)/1O or 2N(Im)/2O atoms in the first Zn shell for the fiber/ ribbon samples (Table S1) To test whether the difference in coordination environments observed in XAS and the associated morphologies may reflect different intra-and inter-sheet Zn 2+ chelation, Ac-Aβ(13-21)H14A (Ac-HAQKLVFFA-NH 2 ) was investigated. As indicated in Figure 1, removal of His14 should eliminate inter-sheet metal binding while preserving coordination along the sheet surface.…”

mentioning

confidence: 99%

“…The intensity and the position of the absorbance at the Zn K-edge region and EXAFS were consistent with four light elements (nitrogen or oxygen) in the coordination sphere of Zn 2+ in the fibers/ribbons (Figure 4a), as well as the supernatant samples. 20,21 Curve fitting indicated either 3N(Im)/1O or 2N(Im)/2O atoms in the first Zn shell for the fiber/ ribbon samples (Table S1) ] samples, could be the result of angular distortion of the imidazoles. 22 Therefore, the local Zn 2+ coordination environment changes coincident with the change in morphology.…”

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confidence: 99%

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Modulating Amyloid Self-Assembly and Fibril Morphology with Zn(II)

et al. 2006

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Interest in the molecular structure of amyloid fibrils originates both from their association with many devastating diseases and as systems for exploring the energetics of higher order protein folding and assembly. These fibril arrays are generally viewed as rich in β-sheets, of either parallel or antiparallel orientation. [1][2][3][4][5] However, the relative arrangement of the sheets within the fibril remains poorly constrained in the existing structure models, 4,6,7 as these sheet-to-sheet arrangements are mediated predominantly by side chain packing. We now extend the use of metal ions as probes of amyloid side chain packing in simple segments of the Aβ peptide of Alzheimer's disease. By restricting the possible metal binding sites, we show that Zn 2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments.The histidine dyad, His13 and His14, of Aβ is implicated in metal binding 8,9 and the metalmediated toxicity of Aβ. [10][11][12][13] In a parallel, in-register β-sheet arrangement with sheet Hbonds oriented along the fibril axis (Figure 1a.), 3,4 the side chains of the His13 and His14 are spaced 5 Å apart along each surface of the β-sheets (Figure 1b). If the sheets are arrayed parallel to one another, the His13 and His14 side chains from different sheets are proximal, providing potential sites for Zn 2+ chelation along the sheets (Figure 1b), between the sheets (Figure 1c), or both. 6 Aβ(13-21), HHQKLVFFA, includes both the core segment, Aβ(17-21), known to be crucial for fibril formation, 14-18 and the metal binding dyad. To isolate His13/14 as the sole binding elements, the K16A peptide HHQALVFFA-NH 2 , Aβ(13-21)K16A, was prepared. As shown in Figure 2a, Aβ(13-21)K16A develops β-sheet secondary structure within 49 h, showing an increased mean residue molar ellipticity (MRME) by circular dichroism (CD) at 197 and 212 nm. The development of β-sheet structure was further confirmed by FTIR, showing the appearance of the amide I absorbance at 1628 cm −1 ( Figure S1a). TEM further established that Aβ(13-21)K16A assembles into fibrils (Fig. S1b), and these mature fibrils bind Congo red with the typical UV/vis absorption shift from 500 to 540 nm ( Figure S1c Figure 3). When the ribbons from the 1:1 incubation were pelleted, washed, and analyzed, the Zn 2+ to peptide ratio was 0.6-0.8 across three independent measurements. 19Wider 100-150 nm ribbons with variable twists form with longer incubation times (panels 2a, b, Figure 3). Some of the ribbons appear to coil and fuse to form tubular structures 200-300 nm in diameter (panels 2c, d, Figure 3). Therefore, Zn 2+ reduces the nucleation time of self-assembly across the entire concentration range and transforms Aβ(13-21)K16A assembly into either fibrillar or ribbon/ tubular morphology. 17 Struck by the different morphologies accessible to Aβ(13-21)-K16A, we investigated the coordination environment of Zn 2+ in the different assemblies by X-ray absorption spectroscopy (XAS). The soluble ...

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confidence: 53%

mentioning

confidence: 99%

“…The intensity and the position of the absorbance at the Zn K-edge region and EXAFS were consistent with four light elements (nitrogen or oxygen) in the coordination sphere of Zn 2+ in the fibers/ribbons (Figure 4a), as well as the supernatant samples. 20,21 Curve fitting indicated either 3N(Im)/1O or 2N(Im)/2O atoms in the first Zn shell for the fiber/ ribbon samples (Table S1) ] samples, could be the result of angular distortion of the imidazoles. 22 Therefore, the local Zn 2+ coordination environment changes coincident with the change in morphology.…”

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confidence: 99%

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Modulating Amyloid Self-Assembly and Fibril Morphology with Zn(II)

et al. 2006

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“…The EXAFS background removal used a three-region, cubic spline. The data were converted to k space by using E 0 ¼ 9664 eV (37), curve fitting details are provided in Table S3 (34). The program Feff v7.02 (38) was used to calculate amplitude and phase functions, for a Zn-O interaction at 2.0 Å, and Zn-P, Zn-C, and Zn-Zn interactions at 3.0 Å.…”

Section: Methodsmentioning

confidence: 99%

“…Data were analyzed in k space by using the program EXAFSPAK (39). For all data, S s was fixed at 0.9 based on fits to the EXAFS data for structurally characterized model complexes (37,40).…”

Section: Methodsmentioning

confidence: 99%

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

Koutmou

Casiano-Negroni

Getz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn 2þ bound to a P4 helix mimic is sixcoordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn 2þ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with CoðNH 3 Þ 3þ 6 , Zn 2þ , and CoðNH 3 Þ 3þ 6 ∕Zn 2þ confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.manganese | ribozyme | RNase P | X-ray absorption spectroscopy | zinc R NA molecules are large polyanions that associate with numerous divalent metal ions that stabilize their structure and promote catalysis. Identification of the RNA moieties involved in metal-binding and discerning the nature of these interactions is an important outstanding question in the field (1, 2). Whereas RNA-bound metals can be characterized at the atomic level in the solid-state by X-crystallography there are not yet techniques for characterizing the binding sites and coordination schemes for RNA-bound metals in solution. Techniques based on NMR, EPR, and Raman spectroscopy as well as nucleotide analog interference mapping (NAIM) and phosphorothioate substitution exist for identifying RNA residues involved in metal-binding; however the geometry of the bound metal and the nature of the coordination scheme cannot be resolved based on these experiments alone.RNase P is a metal-dependent ribozyme that catalyzes precursor tRNA (pre-tRNA) maturation by cleaving a specific phosphodiester bond (3). In RNase P, metal ions stabilize the folded RNase P RNA (PRNA) structure, enhance ligand affinity, and stabilize the transition state for cleavage (4); in vivo the metal requirement is fulfilled by Mg 2þ ions (5, 6). A majority of the ∼150 divalent metal ions associated with PRNA bind nonspecifically via electrostatic interactions whereas only a handful of ions form specific contacts with RNase P (5, 7). Discerning the position and structure of the few divalent ions that site-specifically interact with RNA is a majo...

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