Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.

The enantioselective synthesis of axially chiral biaryls by a copper-catalyzed Diels−Alder/retro-Diels−Alder reaction of 2-pyrones with alkynes is reported herein. Using electron-deficient 2-pyrones and electron-rich 1-naphthyl acetylenes as the reaction partners, a broad range of axially chiral biaryl esters are obtained in excellent yields (up to 97% yield) and enantioselectivities (up to >99% ee). DFT calculations reveal the reaction mechanism and provide insights into the origins of the stereoselectivities. The practicality and robustness of this reaction are showcased by gram-scale synthesis. The synthetic utilizations are demonstrated by the amenable transformations of the products.

The streptavidin–biotin controlled binding probe has several advantages for the detection of enzymes and reactive small molecules, such as minimal background, multiple signal amplification steps, and wide selection of the optimal dyes for detection.

Lateral flow assay (LFA) has been a valuable diagnostic tool in many important fields where rapid, simple, and on-site detection is required, for applications such as pregnancy tests and infectious disease prevention. Currently, two types of LFAs are available: lateral flow immunoassay (LFIA) and nucleic acid lateral flow assay (NALFA). Both are generally used for the testing of proteins and nucleic acids. However, enzyme activities and small molecules without the corresponding binding partner cannot be detected by the existing LFAs. In this paper, we introduce a LFA approach termed affinity-switchable lateral flow assay (ASLFA) to overcome the limitations. The detection principle is based on the switchable binding between the affinity-switchable biotin (ASB) probe and avidin protein. In the presence of the target molecule, the activated ASB probe would be captured by the avidin, thereby leaving a distinct test line on the membrane. The ASLFA concept was demonstrated by testing the F ion, NADH cofactor, and nitroreductase activity. Thus, this general ASLFA can be used for the rapid detection of molecules that cannot be accessed by the classical LFAs.

We are introducing a new approach to evaluate cellular uptake of drugs and drug candidates into living cells. The approach is based on converting the protein target of a given class of compounds into a fluorescent biosensor. By measuring the binding of different compounds to their cognate biosensor in live cells and comparing these values to those measured in vitro, their cellular uptake and concentrations can be ranked. We demonstrate that our strategy enables the evaluation of the cellular uptake into the cytosol of 2 classes of inhibitors using two different sensor designs; first, sensors comprising the self-labeling protein SNAP conjugated with a chemically modified inhibitor shown for inhibitors of the enzyme human carbonic anhydrase II; and a label-free sensor for inhibitors of protein-protein interactions demonstrated for the protein pair p53-HDM2.

Molecular recognition (e.g., antigen–antibody, DNA–DNA, and streptavidin–biotin) is a generic, yet highly versatile and powerful strategy employed in enzyme-catalyzed signal amplification process. However, this approach is not applicable to metals, anions, and small reactive species (e.g., O2 – and F–), as these molecules are too small to bind effectively to the macromolecules. In this paper, we demonstrate an enzyme-catalyzed signal amplification approach based on the controlled binding between streptavidin and target activated affinity-switchable biotin (ASB) probes, for the detection of O2 – and F–, using electrochemical and fluorescent detection techniques. The underlying rationale behind this design is that, while the ASB probe would not bind with the streptavidin–enzyme conjugate due to its low binding affinity with streptavidin, in the presence of the target analyte, the ASB probe on the immobilized surface will be activated to form biotin, which can then bind with the enzyme-tagged streptavidin to initiate signal amplification process. This versatile approach can also be applied in the imaging of endogenously secreted O2 – along the plasma membrane of living cells using streptavidin conjugated with multiple fluorescent dye reporters. We believe that this ASB probe strategy will be useful for a wide range of applications, such as in basic biological research and medical diagnoses, where highly specific signal enhancement is required.

We study the effects of metal-enhanced fluorescence (MEF) on rhodamine B fluorophore by nanoparticles of varied shapes. The avidin−biotin system was used as spacer to connect the fluorophore to the surface of nanoparticles. Fluorescence lifetime image microscopy (FLIM) was used to detect emission lifetime for dye molecules on single nanoparticles. Spherical gold particles diameter of 60 and 170 nm, respectively, cube length of 70 nm, and rhombic dodecahedron (RD) diameter of 63 nm were used. In the measured emission curves of rhodamine B, we obtained a short component with lifetime 16−26 ps attributed to the fluorophore under influence of the local electric field of gold nanoparticle and energy dissipation to nanoparticle. The second lifetime is 200, 270, 280, and 330 ps for 170 nm sphere, 70 nm cube, 63 nm RD, and 60 nm sphere, respectively. This component is referred to as the bright mode of nanoparticle which is coupled to the excited dye molecule and transfers energy back to the fluorophore. On the basis of the large amplitude obtained for the short lifetime component, the effect of MEF was great. The avidin−biotin assembly serves as a biospacer in the MEF applications. Moreover, the MEF effect on the Ag shell gold nanoparticle is studied. Various thicknesses of Ag shell around 40 nm diameter gold core nanoparticles were synthesized. In these Au@Ag−R nanoparticles, time constants obtained in rhodamine B emission curves are τ 1 /τ 2 = 15/210, 23/240, 26/251, 21/246, 25/255 ps for Ag shell thickness of 4, 6, 7.5, 13, and 16 nm, respectively. Because of this multiple exponential decay behavior, we derived a kinetic model for the MEF process and calculated the rate constants of energy transfer between the dye molecules and the Au@Ag nanoparticle. As rhodamine B is excited, it can transfer energy to Au nanoparticle and also dissipates energy to Ag shell via nanosurface energy transfer (NSET), leading to severe fluorescence quenching. This results in low enhancement factors of fluorescence in this core−shell system. According to the experimental lifetime data, the NSET rate constant for the energy dissipation to Ag surface is estimated to be (4−6.6) × 10 10 s −1 .

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