Multimeric Rhodamine Dye-Induced Aggregation of Silver Nanoparticles for Surface-Enhanced Raman Scattering

Isotopic variants of Rhodamine 6G (R6G) have previously been used as a method of multiplexed detection for Surface Enhanced Raman Spectroscopy (SERS), including protein detection and quantification. Challenges exist, however, with producing long-term stable SERS signals with exposure to silver or gold metal surfaces without the use of additional protective coatings of nanomaterials. Here, novel rhodamine “dimers” and “trimers” have been created that demonstrate a higher avidity for metal nanoparticles and induce aggregation to create plasmonic “hotspots” as indicated by enhanced Raman scattering in situ. These aggregates can be formed in a colloid, on surfaces, or membrane substrates such as poly­(vinylidene fluoride) for applications in biosciences. The integrity of the materials and Raman signals are maintained for months of time on different substrates. These dye materials should provide avenues for simplified in situ generation of sensors for Raman-based assays especially in settings requiring highly robust performance

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

<div><p>The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.</p></div

Interactions of 2 and 6 substituted benzopyranone analogs with MBP-HPV-16 E6.

<p>IC50 and maximal inhibition determined in the MBP-HPV-16 E6/E6AP binding assay; ΔTM change of MBP-E6 and MBP at 50 μM compound over DMSO control in the TSA; TSA MBP-E6—apparent <i>K</i>_<i>d</i> of compound interaction; IC50 in the p53 <i>in vitro</i> degradation assay with MBP-E6. N.d. not determined.</p

The rim arginines R10, R55, R102, R129, and R131 play a major role in the shape of the E6 helical binding groove, as well as in molecular recognition of the binding partners.

<p>(A) The arginine residues (yellow-gold color) form multiple polar contacts (blue dotted lines) with E6AP (green backbone), which are proposed to contribute to its binding affinity and alpha-helical shape (B,C). Concurrently, the rim arginines also form important intra-molecular polar contacts (blue doted lines; all HPV-16 E6 residues involved in polar contacts with the rim arginines are colored red), which are believed to shape the floor and sides of the groove. R102 is important for groove floor formation (B), while R131 plays a role in forming the right side of the binding pocket (C).</p

Characterization of wild-type MBP-HPV-16 E6.

<p>(A) Time course and (B) Dose response of MBP-E6 mediated p53 <i>in vitro</i> degradation activity. (C) Binding capacity of wild-type E6 and mutants F2V and L50G to E6AP in the bead-based assay. (D) p53 <i>in vitro</i> degradation with F2V and L50G. (E,F) Thermal stability profiles of MBP, MBP-E6 and MBP-E6 L50G. All data are expressed as S.E.M. *P<0.05.</p

CAF-25 binding to MBP-HPV-16 E6.

<p>(A) Dose response effect of CAF-25 on MBP-E6/E6AP binding in the bead-based assay. (B) Effect of maltose on MBP melt curve in the TSA. (C) Dose dependent effect of maltose on the melting temperature changes (ΔTM) on MBP and MBP-E6. (D) Raw fluorescence plot of MBP-E6 melt curves in response to increasing CAF-25. Relative fluorescence plots of MBP-E6 (E) and MBP (F) melt curves in response to CAF-25. Negative control is composed of Sypro Orange in buffer. (G) Summary of the ΔTM changes of MBP and MBP-E6 by CAF-25. (H) Inhibition of MBP-E6 mediated p53 degradation by CAF-25. All data are expressed as S.E.M. *P<0.05.</p

Interaction of CAF-25 and CAF-26 with MBP-E6 R131A protein.

<p>ΔTM profile of MBP-E6 and R131A in response to increasing concentrations of (A) CAF-25 and (B) CAF-26. (C) Western blot of the p53 <i>in vitro</i> degradation activity of MBP-E6 wild-type (top) and R131A mutant (bottom) in response to CAF-26. p53 protein and MBP-E6 proteins were blotted using p53 (pAB1801) and anti-E6 antibody (AVR 813). (D) Densitometric analysis of p53 protein expressed as fold change over p53 control for wild-type E6 and R131A in response to increasing CAF-26. All data are expressed as S.E.M. * P<0.05.</p

Molecular dynamics (MD) simulations of CAF-25 and CAF-40 with HPV-16 E6 (PDB ID: 4GIZ).

<p>The five arginine residues surrounding the binding groove are highly flexible (A) and both CAF-25 and CAF-40 assume similar docking orientations, maximizing their interactions with R102, R129, and R131 (B). Panels C and D show a detailed interaction map with the binding groove of CAF-25 and CAF-40, respectively. In both cases, H-bonding and π-cation contacts with R102, R129, R131, K11, C51, and S74 form the primary basis for interaction with E6.</p

Characterization of HPV-16 E6 arginine mutants.

<p>(A) Binding of MBP-E6 wild-type and mutants R10A, R55A, R102A and R131A to E6AP in the bead-based assay. (B) p53 <i>in vitro</i> degradation by wild-type and MBP-E6 mutants. Top panel: representative western blots blotted with anti-p53 and anti-E6. Bottom panel: Densitometric analysis of p53 expressed as fold change over p53 control. (C) C33a cells transiently transfected with p53-luc, firefly luciferase and increasing DNA concentrations of 16E6 and mutants analyzed for luciferase activities at 24 hrs. All data are expressed as S.E.M. *P<0.05.</p

The unique topography of the α-helix binding groove of HPV-16 E6 is essential in maintaining strong polar contacts with the E6 binding motif.

<p>The rim arginines <b>R10, R55, R102, R129,</b> and <b>R131</b> form multiple hydrogen bonds with both backbone and side-chain atoms. Other key highlighted residues are <b>K11,</b> and <b>L50</b>, which are proposed to be crucial for maintaining E6 shape and activity.</p