A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins.

p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways.

Importance of polarization effect in the study of metalloproteins: application of polarized protein specific charge scheme in predicting the reduction potential of azurin.

Molecular dynamics (MD) simulation is commonly used in the study of protein dynamics, and in recent years, the extension of MD simulation to the study of metalloproteins is gaining much interest. Choice of force field is crucial in MD studies, and the inclusion of metal centers complicates the process of accurately describing the electrostatic environment that surrounds the redox centre. Herein, we would like to explore the importance of including electrostatic contribution from both protein and solvent in the study of metalloproteins. MD simulations with the implementation of thermodynamic integration will be conducted to model the reduction process of azurin from Pseudomonas aeruginosa. Three charge schemes will be used to derive the partial charges of azurin. These charge schemes differ in terms of the amount of immediate environment, respective to copper, considered during charge fitting, which ranges from the inclusion of copper and residues in the first coordination sphere during density functional theory charge fitting to the comprehensive inclusion of protein and solvent effect surrounding the metal centre using polarized protein-specific charge scheme. From the simulations conducted, the relative reduction potential of the mutated azurins respective to that of wild-type azurin (ΔEcal) were calculated and compared with experimental values. The ΔEcal approached experimental value with increasing consideration of environmental effect hence substantiating the importance of polarization effect in the study of metalloproteins. This study also attests the practicality of polarized protein-specific charge as a computational tool capable of incorporating both protein environment and solvent effect into MD simulations.

Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection?

One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV-Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV-Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.

Cavity-creating mutations in Pseudomonas aeruginosa azurin: effects on protein dynamics and stability.

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (tau(0)) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (k(q)) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations.

Electronic coupling between azurin and gold at different protein/substrate orientations.

By means of constrained classical molecular dynamics simulations, we have computed the structure of azurin deposited on a Au(111) surface at different possible orientations and the azimuthal forces acting on the protein at each sampled conformation. We have then evaluated the effect of the angular variation on the speed of electron tunneling between the protein redox site and the metal surface. We find that the azurin/gold electronic coupling has a strong dependence on the molecular orientation and is greatly enhanced by inclining the protein to lie as flat as possible on the surface. We discuss the implications of our results for scanning probe microscopy experiments in which tunneling currents are measured while the protein is subjected to mechanical forces exerted by the tip of the instrument.

Protein stability in ice.

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

Role of protein cavities on unfolding volume change and on internal dynamics under pressure.

The effects of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (DeltaV(0)) of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift of the center of mass of the fluorescence spectrum, showed that DeltaV(0) is in the range of 60-70 mL/mol, not significantly different between cavity mutants and compact azurin species such as the wild-type and the mutant C3A/C26A, in which the superficial disulphide has been removed. The lack of extra volume in F110S and I7S proves that the engineered cavities, 40 A(3) in I7S and 100 A(3) in F110S, are filled with water molecules. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)). The application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between compact folds, wild-type and C3A/C26A, and cavity mutants is that the inversion point is powered from approximately 3 kbar to 1.5 kbar for F110S and <0.1 kbar for I7S, meaning that in the latter species pressure-induced internal hydration dominates very early over any compaction of the globular fold resulting from the reduction of internal free volume. The similar response between wild-type and the significantly less-stable C3A/C26A mutant suggests that thermodynamic stability per se is not the dominant factor regulating pressure-induced internal hydration of proteins.

Solid state protein monolayers: morphological, conformational, and functional properties.

We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

Real-time scanning tunnelling microscopy imaging of protein motion at electrode surfaces.

A mutant (K27C) of the blue copper protein azurin [Eur. J. Biochem. 194 (1990) 109; J. Mol. Biol. 221 (1991) 765] for orientated immobilisation on gold surfaces was analysed by scanning tunnelling microscopy (STM) both in a resting state and following the application of a short potential pulse between the tip and sample.

An approach to long-range electron transfer mechanisms in metalloproteins: in situ scanning tunneling microscopy with submolecular resolution.

In situ scanning tunneling microscopy (STM) of redox molecules, in aqueous solution, shows interesting analogies and differences compared with interfacial electrochemical electron transfer (ET) and ET in homogeneous solution. This is because the redox level represents a deep indentation in the tunnel barrier, with possible temporary electronic population. Particular perspectives are that both the bias voltage and the overvoltage relative to a reference electrode can be controlled, reflected in spectroscopic features when the potential variation brings the redox level to cross the Fermi levels of the substrate and tip. The blue copper protein azurin adsorbs on gold(111) via a surface disulfide group. Well resolved in situ STM images show arrays of molecules on the triangular gold(111) terraces. This points to the feasibility of in situ STM of redox metalloproteins directly in their natural aqueous medium. Each structure also shows a central brighter contrast in the constant current mode, indicative of 2- to 4-fold current enhancement compared with the peripheral parts. This supports the notion of tunneling via the redox level of the copper atom and of in situ STM as a new approach to long-range electron tunneling in metalloproteins.

Conformational substates in azurin.

Azurin is a small blue copper protein in the electron transfer chain of denitrifying bacteria. It forms a photolabile complex with nitric oxide (NO) at low temperatures. We studied the temperature dependence of the ligand binding equilibrium and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). The nonexponential rebinding below 200 K is independent of the NO concentration and is interpreted as internal recombination. The rebinding can be modeled with the Arrhenius law by using a single preexponential factor of 6.3 x 10(8) s-1 and a Gaussian distribution of enthalpy barriers centered at 23 kJ/mol with a width of 11 kJ/mol. Above 200 K, a slower, exponential rebinding process appears. The dependence of the kinetics on the NO concentration characterizes this reaction as bimolecular rebinding. The binding kinetics of NO to azurin show impressive analogies to the binding of carbon monoxide to myoglobin. We conclude that conformational substates occur not only in heme proteins but also in proteins with different active sites and secondary structures.

EXAFS of the type-1 copper site of rusticyanin.

Extended X-ray absorption fine structure (EXAFS) spectra at the Cu K-edge have been recorded of the oxidized and reduced form at pH 3.5 of rusticyanin, the type-1 or 'blue'-copper protein from Thiobacillus ferrooxidans. The EXAFS of oxidized rusticyanin is well simulated with models assuming a ligand set of 2 N(His) and 1 S(Cys) at 1.99 and 2.16 A, respectively. Upon reduction, the average Cu-N ligand distance increases by approx. 0.08A. For both redox states studied, the fit by the simulation is significantly improved by including a contribution of an additional sulfur ligand at approx. 2.8 A. From comparison with structural data of other blue-copper proteins, it is concluded that the copper coordination environment is relatively rigid, which may be a clue to its high redox potential.