Genetic engineering of a novel protein–nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system ...is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18‐subunit double ring, which self‐assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His‐β‐loopless, HBLL) with increased access to the central cavity and His‐tags on each subunit extending into the central cavity. This mutant binds water‐soluble semiconductor quantum dots, creating a protein‐encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody–antigen interactions, a one‐to‐one protein–nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent‐exposed regions of the chaperonin, this protein–nanoparticle bioconjugate becomes a sensor for specific targets.
A mutant chaperonin originating from S. Shibatae is genetically engineered to bind water‐soluble semiconductor quantum dots, forming a bioconjugate characterized by high stability, controlled stoichiometry, enhanced photostability, and brightness of the bound quantum dots (see image). This bioconjugate becomes a highly specific biosensor for biological targets when ligands are inserted at the protein solvent exposed side.
Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence ...self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.
Biotin was covalently tagged with a BODIPY dye which can undergo an efficient distance-dependent fluorescence self-quenching. Multivalent binding of avidin with the BODIPY-labeled biotin (B
...581/591-biotin, either in aqueous buffer, or anchored on the surfaces of lipid vesicles or lipid bilayers coated on glass beads) induces aggregation of the BODIPY dye (up to four dyes for each avidin) to result in a decrease in fluorescence intensity due to fluorescence self-quenching. The system can be used to perform a rapid, direct assay for avidin and competitive assay for biotin with high sensitivity (<50
pM for avidin and <0.2
nM for biotin) and selectivity. The assay method is generally applicable for detection of all the species involved in a multivalent binding interaction.
We have developed a novel approach for simple and convenient detection of ligand−receptor interactions with high sensitivity and selectivity. The method harnesses a reversible fluorescence quenching ...between nonfluorescent polyelectrolyte quenchers and fluorescent probes tagged to ligands. Formation of more strongly bound ligand−receptor complexes forces the breakup of nonspecific, electrostatic interaction-based quencher/probe pairs to reverse the fluorescence quenching, triggering a fluorescence increase, and providing an off-to-on detection of the ligand/receptor interaction. Although a conjugated polyelectrolyte was used in the study, other nonconjugated polyelectrolyte quenchers are expected to work as well. The method should potentially find a wide variety of applications in several areas, ranging from drug screening, medical diagnostics, and biosensing to assay development.
Vibrational data (IR, Raman and inelastic neutron scattering) and a supporting normal coordinate analysis for the complex trans-W(CO)3(PCy3)2(η2-H2) (1) and its HD and D2 isotopomers are reported. ...The vibrational data and force constants support the well-established η2-bonding mode for the H2 ligand and provide unambiguous assignments for all metal−hydrogen stretching and bending frequencies. The force constant for the HH stretch, 1.3 mdyn/Å, is less than one-fourth the value in free H2 and is similar to that for the WH stretch, indicating that weakening of the H−H bond and formation of W−H bonds are well along the reaction coordinate to oxidative addition. The equilibrium isotope effect (EIE) for the reversible binding of dihydrogen (H2) and dideuterium (D2) to 1 and 1-d 2 has been calculated from measured vibrational frequencies for 1 and 1-d 2. The calculated EIE is “inverse” (1-d 2 binds D2 better than 1 binds H2), with K H/K D = 0.78 at 300 K. The EIE calculated from vibrational frequencies may be resolved into a large normal mass and moment of inertia factor (MMI = 5.77), an inverse vibrational excitation factor (EXC= 0.67), and an inverse zero-point energy factor (ZPE = 0.20), where EIE = MMI × EXC × ZPE. An analysis of the zero-point energy components of the EIE shows that the large decrease in the HH stretching frequency (force constant) predicts a large normal EIE but that zero-point energies from five new vibrational modes (which originate from translational and rotational degrees of freedom from hydrogen) offset the change in zero-point energy from the H2(D2) stretch. The calculated EIE is compared to experimental data obtained for the binding of H2 or D2 to Cr(CO)3(PCy3)2 over the temperature range 12−36 °C in THF solution. For the binding of H2 ΔH = −6.8 ± 0.5 kcal mol-1 and ΔS = −24.7 ± 2.0 cal mol-1 deg-1; for D2 ΔH = −8.6 ± 0.5 kcal/mol and ΔS = −30.0 ± 2.0 cal/(mol deg). The EIE at 22 °C has a value of K H/K D = 0.65 ± 0.15. Comparison of the equilibrium constants for displacement of N2 by H2 or D2 in the complex W(CO)3(PCy3)2(N2) in THF yielded a value of K H/K D = 0.70 ± 0.15 at 22 °C.
Using temperature-dependent Fourier transform infrared (FTIR) spectroscopy, we probe the molecular level, chain-structural dynamics associated with solid−solid transitions between 25 and 250 °C in a ...layered inorganic−organic silver dodecanethiolate, AgS(CH2)11CH3. Spectroscopic evidence presented here establishes two major transitions: the transition occurring at ∼130 °C is characterized by an abrupt, but fully reversible, change in the chain conformational order from an initial all-trans state to the one characterized by mixed or partial chain disorder. The observation of this phase transition is consistent with the previous predictions of a rapid and drastic change in the structural motif from an initial bilayer to the final micellar state. The second transition at about 190 °C, which is consistent with the previous assignment of micellar amorphous transition, is furthermore irreversible and represents thermal degradation of the material. Implications of these results for the general family of chain molecular assemblies in constrained molecular environments are discussed.
The synthesis and characterization of covalently bound self-assembled monolayer thin films of 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (TPyP) and its derivatives on fused quartz and silicon 100 ...substrates having a native oxide layer are described. The monolayer film consists of porphyrin macrocycle disk-like structures that were analyzed by UV-visible spectroscopy, X-ray photoelectron spectroscopy (XPS), and polarized FTIR-ATR measurements. One of the attractive features of these complexes is their large second-order nonlinear optical response, as expected for a strongly delocalized pi-electron system without inversion symmetry. Second-harmonic generation (SHG) measurements have been used to determine the absolute value of the dominant element of the nonlinear susceptibility, chi-zzz approximately 2 times 10sup minus8 esu, the uniformity of these films, and the dispersion of the refractive indices. The average molecular orientation angle of the surface-bound chromophores was measured by both FTIR-ATR and SHG and found to be in good agreement. 31 refs., 3 figs.
The binding properties of cholera toxin B (CTB) oligomer to substrate supported membrane bilayer, containing physiologically relevant concentrations of receptor glycolipids,
viz. monosialoganglioside ...(GM1), have been extensively studied by the atomic force microscopy (AFM). Two distinct classes of GM1 containing membrane-mimetic surfaces were prepared: supported lipid bilayer membranes (sBLMs) on freshly cleaved mica and hybrid lipid bilayer membranes (hBLMs) on octadecyltrichlorosilane (OTS) derivatized silicon substrates. On sBLMs, aggregates with a well-defined ordered arrangement of individual CTB molecules were observed at all GM1 and cholera concentrations studied. In sharp contrast, features consistent with randomly distributed adsorbed individual CTB molecules were seen on a bare mica surface. On the hBLMs, the aggregate structures were only observed when the bilayer was formed onto ordered OTS surfaces, offering continuous and defect-free lipid membrane for the lateral diffusion of GM1. Ill-packed and disordered OTS monolayers yielded a random distribution of adsorbed proteins comparable to that observed for CTB binding on bare mica substrates. These observations strongly support that the aggregation of CTB–GM1 complex is a result of the specific interaction of CTB molecules with GM1 receptors in the fluid membrane bilayers. The high mobility of GM1 allows lateral diffusion of the complex to form ordered aggregates.
