The growing interest in recent years in gold island films prepared by vapor deposition on transparent substrates is largely attributed to the prominent localized surface plasmon (SP) extinction associated with nanostructured metal films. In the present study, two types of evaporated Au island films were investigated: (i) Au films (2.5, 5.0, and 7.5 nm nominal thickness) evaporated on silanized glass and annealed 20 h at a temperature <250 °C; (ii) Au films (7.5 and 10 nm nominal thickness) evaporated on unmodified glass and annealed 10 h at 550 or 600 °C. The 3D morphology of the Au islands was analyzed using high-resolution scanning electron microscopy (HRSEM), crosssectional transmission electron microscopy (TEM), and atomic force microscopy (AFM) crosssectional profilometry. Annealing at high temperatures, close to the glass transition temperature of the substrate, results in wetting of the Au islands by the glass and partial island embedding. The mechanism of morphology evolution during annealing changes from island coalescence and coarsening (low nominal thicknesses) to dewetting of percolated films (higher nominal thicknesses). The aspect ratio of more than 90% of the islands in annealed films is <1.5; therefore, splitting of the SP band to transversal and longitudinal components is not observed. The bulk refractive index sensitivity (RIS), in terms of SP wavelength shift and plasmon intensity change (PIC) per refractive index unit (RIU) change of the medium, was determined by measuring UV-vis spectra of Au island films in a series of methanol/chloroform mixtures. The RIS values for SP wavelength shift (RIS λ ) and PIC (RIS ext ) are 66-153 nm/RIU and 0.2-0.81 abs.u./RIU, respectively. The RIS shows a strong dependence on the wavelength of the SP maximum extinction, i.e., a higher RIS is measured for Au island films exhibiting a SP band at longer wavelengths. Partial thermal embedding of the Au islands in the glass substrate stabilizes the systems but lowers the RIS. The results presented may be useful for tuning the morphology and optical response of Au island films.

Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.allostery | protein dynamics | single-molecule enzymology

The molecular chaperone GroEL exists in at least two allosteric states, T and R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, ∼50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.allostery | conformational dynamics | fluorescence correlation spectroscopy | molecular chaperones | chaperonins A TP-driven protein machines are abundant and contribute to multiple essential biological processes (1). Such protein machines undergo motor-like rotational motion (like F 1 -ATPase), carry cargos (like kinesin), or fold proteins (like GroEL, the subject of this paper). An important feature of all ATP-driven molecular machines is a functional cycle that involves sequential transitions between several conformational states (2). Understanding the dynamics of conformational cycling is, therefore, essential for the full elucidation of the mechanism of action of a protein machine.The Escherichia coli molecular chaperone GroEL is a machine that assists protein folding by undergoing a series of allosteric transitions that facilitate protein substrate binding and release (3,4). GroEL is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end (5), in which protein folding can take place. The allosteric transitions of GroEL are induced by ATP binding that occurs with positive cooperativity within rings and negative cooperativity between rings (6). It has been suggested that the intraring positive cooperativity is an outcome of a concerted switch between two conformations, Tand R, with low and high affinities for ATP, respectively. GroEL functions in conjunction with a heptameric ring-shaped cochaperonin, GroES. Binding of GroES to the so-called cis ring induces an additional conformational change that leads to the R' conformation and triggers dissociation of bound protein substrate into the cavity (7). The structures of the three relatively stable conformations, T, R, and R', have been determined using x-ray crystallography and electron microscopy (8, 9). It is not known ...