Conformational Heterogeneity and Frustration of the Tumor Suppressor p53 as Tuned by Punctual Mutations.

The conformational heterogeneity of the p53 tumor suppressor, the wild-type (p53wt) and mutated forms, was investigated by a computational approach, including the modeling and all atoms of the molecular dynamics (MD) simulations. Four different punctual mutations (p53R175H, p53R248Q, p53R273H, and p53R282W) which are known to affect the DNA binding and belong to the most frequent hot-spot mutations in human cancers, were taken into consideration. The MD trajectories of the wild-type and mutated p53 forms were analyzed by essential dynamics to extract the relevant collective motions and by the frustration method to evaluate the degeneracy of the energy landscape. We found that p53 is characterized by wide collective motions and its energy landscape exhibits a rather high frustration level, especially in the regions involved in the binding to physiological ligands. Punctual mutations give rise to a modulation of both the collective motions and the frustration of p53, with different effects depending on the mutation. The regions of p53wt and of the mutated forms characterized by a high frustration level are also largely involved in the collective motions. Such a correlation is discussed also in connection with the intrinsic disordered character of p53 and with its central functional role.

Interaction of miR-155 with Human Serum Albumin: An Atomic Force Spectroscopy, Fluorescence, FRET, and Computational Modelling Evidence.

This study investigated the interaction between Human Serum Albumin (HSA) and microRNA 155 (miR-155) through spectroscopic, nanoscopic and computational methods. Atomic force spectroscopy together with static and time-resolved fluorescence demonstrated the formation of an HSA/miR-155 complex characterized by a moderate affinity constant (KA in the order of 104 M-1). Förster Resonance Energy Transfer (FRET) experiments allowed us to measure a distance of (3.9 ± 0.2) nm between the lone HSA Trp214 and an acceptor dye bound to miR-155 within such a complex. This structural parameter, combined with computational docking and binding free energy calculations, led us to identify two possible models for the structure of the complex, both characterized by a topography in which miR-155 is located within two positively charged pockets of HSA. These results align with the interaction found for HSA and miR-4749, reinforcing the thesis that native HSA is a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence, FRET, Atomic Force Spectroscopy and Computational Modelling.

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M-1 has been assessed through a Stern-Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.

Solution structure of the anticancer p28 peptide in biomimetic medium.

The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We have studied its structure in water and in a biomimetic media and show that the peptide is unstructured in water but when studied in a biomimetic medium assumes a structure very similar to the one observed in azurin, suggesting a high propensity of this peptide to maintain this secondary structure. Analysis of p28 sequences from different bacterial species indicates conservation of the secondary structure despite amino acid replacement in different positions, suggesting that others, similar peptides could be tested for binding to p53.

Toward Cancer Diagnostics of the Tumor Suppressor p53 by Surface Enhanced Raman Spectroscopy.

The tumor suppressor p53 protein plays a crucial role in many biological processes. The presence of abnormal concentrations of wild-type p53, or some of its mutants, can be indicative of a pathological cancer state. p53 represents therefore a valuable biomarker for tumor screening approaches and development of suitable biosensors for its detection deserves a high interest in early diagnostics. Here, we revisit our experimental approaches, combining Surface Enhanced Raman Spectroscopy (SERS) and nanotechnological materials, for ultrasensitive detection of wild-type and mutated p53, in the perspective to develop biosensors to be used in clinical diagnostics. The Raman marker is provided by a small molecule (4-ATP) acting as a bridge between gold nanoparticles (NPs) and a protein biomolecule. The Azurin copper protein and specific antibodies of p53 were used as a capture element for p53 (wild-type and its mutants). The developed approaches allowed us to reach a detection level of p53 down to 10-17 M in both buffer and serum. The implementation of the method in a biosensor device, together with some possible developments are discussed.

A Reliable BioFET Immunosensor for Detection of p53 Tumour Suppressor in Physiological-Like Environment.

The concentration of wild-type tumour suppressor p53wt in cells and blood has a clinical significance for early diagnosis of some types of cancer. We developed a disposable, label-free, field-effect transistor-based immunosensor (BioFET), able to detect p53wt in physiological buffer solutions, over a wide concentration range. Microfabricated, high-purity gold electrodes were used as single-use extended gates (EG), which avoid direct interaction between the transistor gate and the biological solution. Debye screening, which normally hampers target charge effect on the FET gate potential and, consequently, on the registered FET drain-source current, at physiological ionic strength, was overcome by incorporating a biomolecule-permeable polymer layer on the EG electrode surface. Determination of an unknown p53wt concentration was obtained by calibrating the variation of the FET threshold voltage versus the target molecule concentration in buffer solution, with a sensitivity of 1.5 ± 0.2 mV/decade. The BioFET specificity was assessed by control experiments with proteins that may unspecifically bind at the EG surface, while 100pM p53wt concentration was established as limit of detection. This work paves the way for fast and highly sensitive tools for p53wt detection in physiological fluids, which deserve much interest in early cancer diagnosis and prognosis.

Probing direct interaction of oncomiR-21-3p with the tumor suppressor p53 by fluorescence, FRET and atomic force spectroscopy.

miRNA-21-3p is overexpressed in a number of cancers and contributes to their development with a concomitant inhibition of the p53 onco-suppressive function. While a direct interaction of p53 with some miRNA precursors (namely pri-miRNAs and pre-miRNAs) was found, no interaction with mature micro RNA has been so far evidenced. It could therefore be very interesting to investigate if a direct interaction of miR-21-3p and p53 is occurring with possible impairment of the p53 onco-suppressive function. Fluorescence and Atomic Force Spectroscopy (AFS) were applied to study the interaction of p53 DNA Binding Domain (DBD) and miRNA-21-3p. Förster resonance energy transfer (FRET) was used to measure the distance between the DBD lone tryptophan (FRET donor) and a dye (FRET acceptor) bound to miRNA-21-3p. AFS and Fluorescence evidenced a direct interaction between miRNA-21-3p and DBD; with the formed complex being characterized by an affinity of 105 M, with a lifetime in the order of seconds. FRET allowed to determine an average distance of 4.0 nm between the DBD lone Trp146 and miRNA-21-3p; consistently with the involvement of the DBD L3 loop and/or the H1 helix in the complex formation, directly involved in the oligomerization and DNA binding. This may suggest that a functional inhibition of p53 could arise from its interaction with the oncogenic miRNA. Evidence of DBD-miRNA-21-3p complex formation may deserve some interest for inspiring novel therapeutic strategies.

Raman Evidence of p53-DBD Disorder Decrease upon Interaction with the Anticancer Protein Azurin.

Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. The Raman spectra of the DBD and Azurin, isolated in solution or forming a complex, were analyzed by a combined analysis based on peak inspection, band convolution, and principal component analysis (PCA). In particular, our attention was focused on the Raman peaks of Tyrosine and Tryptophan residues, which are diagnostic markers of protein side chain environment, and on the Amide I band, of which the deconvolution allows us to extract information about α-helix, ß-sheet, and random coil contents. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin. These findings suggest an Azurin-induced conformational change of DBD structure with possible implications for p53 functionality.

Structure, Dynamics, and Electron Transfer of Azurin Bound to a Gold Electrode.

Blue copper redox protein azurin (AZ) constitutes an ideal active element for building bionano-optoelectronic devices based on the intriguing interplay among its electron transfer (ET), vibrational, and optical properties. A full comprehension of its dynamical and functional behavior is required for efficient applications. Here, AZ bound to gold electrode via its disulfide bridge was investigated by a molecular dynamics simulation approach taking into account for gold electron polarization which provides a more realistic description of the protein-gold interaction. Upon binding to gold, AZ undergoes slight changes in its secondary structure with the preservation of the copper-containing active site structure. Binding of AZ to gold promotes new collective motions, with respect to free AZ, as evidenced by essential dynamics. Analysis of the ET from the AZ copper ion to the gold substrate, performed by the Pathways model, put into evidence the main residues and structural motifs of AZ involved in the ET paths. During the dynamical evolution of the bionanosystem, transient contacts between some lateral protein atoms and the gold substrate occurred; concomitantly, the opening of additional ET channels with much higher rates was registered. These results provide new and detailed insights on the dynamics and ET properties of the AZ-gold system, by also helping to rationalize some imaging and conductive experimental evidences and also to design new bionanodevices with tailored features.

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman, Atomic Force and Surface Plasmon Resonance spectroscopies.

BACKGROUND: Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. METHODS: Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. RESULTS: We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the ß-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. CONCLUSIONS: These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. GENERAL SIGNIFICANCE: Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.

Surface Plasmon Resonance Sensing of Biorecognition Interactions within the Tumor Suppressor p53 Network.

Surface Plasmon Resonance (SPR) is a powerful technique to study the kinetics of biomolecules undergoing biorecognition processes, particularly suited for protein-protein interactions of biomedical interest. The potentiality of SPR was exploited to sense the interactions occurring within the network of the tumor suppressor p53, which is crucial for maintaining genome integrity and whose function is inactivated, mainly by down regulation or by mutation, in the majority of human tumors. This study includes p53 down-regulators, p53 mutants and also the p53 family members, p63 and p73, which could vicariate p53 protective function. Furthermore, the application of SPR was extended to sense the interaction of p53 with anti-cancer drugs, which might restore p53 function. An extended review of previous published work and unpublished kinetic data is provided, dealing with the interaction between the p53 family members, or their mutants and two anticancer molecules, Azurin and its cell-penetrating peptide, p28. All the kinetic results are discussed in connection with those obtained by a complementary approach operating at the single molecule level, namely Atomic Force Spectroscopy and the related literature data. The overview of the SPR kinetic results may significantly contribute to a deeper understanding of the interactions within p53 network, also in the perspective of designing suitable anticancer drugs.

Energy landscape investigation by wavelet transform analysis of atomic force spectroscopy data in a biorecognition experiment.

Force fluctuations recorded in an atomic force spectroscopy experiment, during the approach of a tip functionalized with biotin towards a substrate charged with avidin, have been analyzed by a wavelet transform. The observation of strong transient changes only when a specific biorecognition process between the partners takes place suggests a drastic modulation of the force fluctuations when biomolecules recognize each other. Such an analysis allows to investigate the peculiar features of a biorecognition process. These results are discussed in connection with the possible role of energy minima explored by biomolecules during the biorecognition process.

Interaction of mutant p53 with p73: a Surface Plasmon Resonance and Atomic Force Spectroscopy study.

BACKGROUND: TP53 tumor suppressor gene is mutated in more than 50% of human tumors. Mutated p53 proteins could sequestrate and inactivate p73 reducing the apoptotic and anti-proliferative effects of the transcription factor, and yielding cancer cells more aggressive and chemoresistant. The possibility of using drugs to prevent the mutant p53/p73 complex formation preserving the p73 function, calls for a deeper insight into the molecular and biochemical mechanisms of mutant p53/p73 protein interaction. METHODS: The kinetics of the mutant p53R175H/p73 complex was investigated with innovative and complementary techniques, operating in real time, in near physiological conditions and without any labeling. Specifically, Atomic Force Spectroscopy and Surface Plasmon Resonance working at single-molecule level and in bulk condition, respectively, were used. RESULTS: The two techniques revealed that a stable complex is formed between mutant p53R175H and p73 proteins; the complex being characterized by a high interaction force and a dissociation equilibrium constant in the order of 10(-7)M, as expected for specific interactions. No binding was instead observed between p73 and wild type p53. CONCLUSIONS: Mutant p53R175H protein, unlike wild type p53, can form a stable complex with p73. The mutant p53R175H/p73 protein complex could be a target for innovative pharmaceutical drugs that, by dissociating it or preventing biomolecule interaction thus preserving the p73 function, could enhance the response of cancerous cells carrying mutant p53R175H protein to common chemotherapeutic agents. GENERAL SIGNIFICANCE: The kinetic information obtained in vitro may help to design specific pharmaceutical drugs directed against cancerous cells carrying mutant p53 proteins.

Chirality switching within an anionic cell-penetrating peptide inhibits translocation without affecting preferential entry.

Multiple substitution of d- for l-amino acids decreases the intracellular uptake of cationic cell penetrating peptides (CPP) in a cell line-dependent manner. We show here that a single d-amino acid substitution can decrease the overall uptake of the anionic, amphipathic CPP, p28, into cancer and histologically matched normal cell lines, while not altering the preferential uptake of p28 into cancer cells. The decrease appears dependent on the position of the d-substitution within the peptide and the ability of the substituted d-amino acid to alter chirality. We also suggest that when d-substitution alters the ratio of α-helix to ß-sheet content of an anionic CPP, its translocation across the cell membrane is altered, reducing overall entry. These observations may have a significant effect on the design of future d-substituted analogues of cell penetrating peptides.

Binding of azurin to cytochrome c 551 as investigated by surface plasmon resonance and fluorescence.

The interaction between azurin (Az) and cytochrome c 551 (CytC551) from Pseudomonas aeruginosa deserves particular interest for both its physiological aspects and their possible applications in bionano devices. Here, the kinetics of the interaction has been studied by surface plasmon resonance and fluorescence quenching. Surface plasmon resonance data have been successfully interpreted by the heterogeneous ligand model, which predicts the existence of two binding sites on the immobilized Az for CytC551 molecules in solution. On the other hand, the fluorescence study indicates the formation of a complex, with the involvement of the lone Az tryptophan (Trp) at position 48. The two different techniques point out the occurrence of an encounter complex between Az and CytC551 that evolves toward the formation of a more stable complex characterized by an equilibrium dissociation constant KD typical of transient interactions.

Antigen-antibody biorecognition events as discriminated by noise analysis of force spectroscopy curves.

Atomic force spectroscopy is able to extract kinetic and thermodynamic parameters of biomolecular complexes provided that the registered unbinding force curves could be reliably attributed to the rupture of the specific complex interactions. To this aim, a commonly used strategy is based on the analysis of the stretching features of polymeric linkers which are suitably introduced in the biomolecule-substrate immobilization procedure. Alternatively, we present a method to select force curves corresponding to specific biorecognition events, which relies on a careful analysis of the force fluctuations of the biomolecule-functionalized cantilever tip during its approach to the partner molecules immobilized on a substrate. In the low frequency region, a characteristic 1/f (α) noise with α equal to one (flickering noise) is found to replace white noise in the cantilever fluctuation power spectrum when, and only when, a specific biorecognition process between the partners occurs. The method, which has been validated on a well-characterized antigen-antibody complex, represents a fast, yet reliable alternative to the use of linkers which may involve additional surface chemistry and reproducibility concerns.

Detection of miR-155 Using Peptide Nucleic Acid at Physiological-like Conditions by Surface Plasmon Resonance and Bio-Field Effect Transistor.

MicroRNAs are small ribonucleotides that act as key gene regulators. Their altered expression is often associated with the onset and progression of several human diseases, including cancer. Given their potential use as biomarkers, there is a need to find detection methods for microRNAs suitable for use in clinical setting. Field-effect-transistor-based biosensors (bioFETs) appear to be valid tools to detect microRNAs, since they may reliably quantitate the specific binding between the immobilized probe and free target in solution through an easily detectable electrical signal. We have investigated the detection of human microRNA 155 (miR-155) using an innovative capturing probe constituted by a synthetic peptide nucleic acid (PNA), which has the advantage to form a duplex even at ionic strengths approaching the physiological conditions. With the aim to develop an optimized BioFET setup, the interaction kinetics between miR-155 and the chosen PNA was preliminarily investigated by using surface plasmon resonance (SPR). By exploiting both these results and our custom-made bioFET system, we were able to attain a low-cost, real-time, label-free and highly specific detection of miR-155 in the nano-molar range.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

Detection of persistent organic pollutants binding modes with androgen receptor ligand binding domain by docking and molecular dynamics.

BACKGROUND: Persistent organic pollutants (POPs) are persistent in the environment after release from industrial compounds, combustion productions or pesticides. The exposure of POPs has been related to various reproductive disturbances, such as reduced semen quality, testicular cancer, and imbalanced sex ratio. Among POPs, dichlorodiphenyldichloroethylene (4,4'-DDE) and polychlorinated biphenyls (PCBs) are the most widespread and well-studied compounds. Recent studies have revealed that 4,4'-DDE is an antagonist of androgen receptor (AR). However, the mechanism of the inhibition remains elusive. CB-153 is the most common congener of PCBs, while the action of CB-153 on AR is still under debate. RESULTS: Molecular docking and molecular dynamics (MD) approaches have been employed to study binding modes and inhibition mechanism of 4,4'-DDE and CB-153 against AR ligand binding domain (LBD). Several potential binding sites have been detected and analyzed. One possible binding site is the same binding site of AR natural ligand androgen 5α-dihydrotestosterone (DHT). Another one is on the ligand-dependent transcriptional activation function (AF2) region, which is crucial for the co-activators recruitment. Besides, a novel possible binding site was observed for POPs with low binding free energy with the receptor. Detailed interactions between ligands and the receptor have been represented. The disrupting mechanism of POPs against AR has also been discussed. CONCLUSIONS: POPs disrupt the function of AR through binding to three possible biding sites on AR/LBD. One of them shares the same binding site of natural ligand of AR. Another one is on AF2 region. The third one is in a cleft near N-terminal of the receptor. Significantly, values of binding free energy of POPs with AR/LBD are comparable to that of natural ligand androgen DHT.

1/fα noise in the dynamic force spectroscopy curves signals the occurrence of biorecognition.

Biorecognition leads to the formation of a specific complex between a couple of biological partners to accomplish a functional task. Its occurrence can be inferred a posteriori by analyzing the unbinding force curves in a dynamic force spectroscopy experiment. Because of nonspecific interactions, the method is not, however, exempt from ambiguities and subjectivity. A fingerprint of the partner recruitment in the complex has been disclosed in the fluctuations of the atomic force microscopy cantilever. We demonstrate that the formation of the biotin-avidin specific complex strongly correlates with a 1/f(α) noise in the force curve fluctuations.