Keywords: Aggregation, Metal-Protein Interactions, Ubiquitin, Zinc, Affinity Constants, Binding Modes, Copper Binding, Dynamic Equilibria, Equimolar Addition, In-Line, Neurodegeneration, Neutral Ph, Potentiometric Titrations, Preferential Binding, Protein Assembly, Protein Stability, Protein Thermal Stability, Ubiquitin-Proteasome System, Ubiquitinated Proteins, Zinc Complex, Agglomeration, Binding Energy, Binding Sites, Complexation, Differential Scanning Calorimetry, Metal Ions, Zinc Compounds, Metalloprotein, Article, Chemistry, Metabolism, Nuclear Magnetic Resonance Spectroscopy, Protein Binding,
Affiliations: *** IBB - CNR ***
Dipartimento di Scienze Chimiche, Universita degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Istituto di Biostrutture e Bioimmagini-UOS CT, Consiglio Nazionale Delle Ricerche, V.le A. Doria 6, 95125 Catania, Italy
References: Not available.
Zinc(II) complexes of ubiquitin: speciation, affinity and binding features
Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Calpha and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Zinc(II) complexes of ubiquitin: speciation, affinity and binding features