Intersubband Polariton-Polariton Scattering in a Dispersive Microcavity.

The ultrafast scattering dynamics of intersubband polaritons in dispersive cavities embedding GaAs/AlGaAs quantum wells are studied directly within their band structure using a noncollinear pump-probe geometry with phase-stable midinfrared pulses. Selective excitation of the lower polariton at a frequency of ∼25 THz and at a finite in-plane momentum k_{‖} leads to the emergence of a narrowband maximum in the probe reflectivity at k_{‖}=0. A quantum mechanical model identifies the underlying microscopic process as stimulated coherent polariton-polariton scattering. These results mark an important milestone toward quantum control and bosonic lasing in custom-tailored polaritonic systems in the mid and far infrared.

Interferometric control of absorption in thin plasmonic metamaterials: general two port theory and broadband operation.

In order to extend the Coherent Perfect Absorption (CPA) phenomenology to broadband operation, the interferometric control of absorption is investigated in two-port systems without port permutation symmetry. Starting from the two-port theory of CPA treated within the Scattering Matrix formalism, we demonstrate that for all linear two-port systems with reciprocity the absorption is represented by an ellipse as function of the relative phase and intensity of the two input beams, and it is uniquely determined by the device single-beam reflectance and transmittance, and by the dephasing of the output beams. The basic properties of the phenomenon in systems without port permutation symmetry show that CPA conditions can still be found in such asymmetric devices, while the asymmetry can be beneficial for broadband operation. As experimental proof, we performed transmission measurements on a metal-semiconductor metamaterial, employing a Mach-Zehnder interferometer. The experimental results clearly evidence the elliptical feature of absorption and trace a route towards broadband operation.

A ballistic quantum ring Josephson interferometer.

We report the realization of a ballistic Josephson interferometer. The interferometer is made from a quantum ring etched in a nanofabricated two-dimensional electron gas confined in an InAs-based heterostructure laterally contacted to superconducting niobium leads. The Josephson current flowing through the structure shows oscillations with h/e flux periodicity when threading the loop with a perpendicular magnetic field. This periodicity, in sharp contrast with the h/2e one observed in conventional dc superconducting quantum interference devices, confirms the ballistic nature of the device in agreement with theoretical predictions. This system paves the way for the implementation of interferometric Josephson π-junctions, and for the investigation of Majorana fermions.

Ordered systems of site-controlled pyramidal quantum dots incorporated in photonic crystal cavities.

The coupling of a prescribed number of site-controlled pyramidal quantum dots (QDs) with photonic crystal (PhC) cavities was studied by polarization and power-dependent photoluminescence measurements. The energy of the cavity mode could be readily tuned, making use of the high spectral uniformity of the QDs and designing PhC cavities with different hole radii. Efficient coupling of the PhC cavity modes both to the ground state and to the excited state transitions of the QDs was observed, whereas no evidence for far off-resonant coupling was found.

Energy transport by neutral collective excitations at the quantum Hall edge.

We use the edge of the quantum Hall sample to study the possibility for counterpropagating neutral collective excitations. A novel sample design allows us to independently investigate charge and energy transport along the edge. We experimentally observe an upstream energy transfer with respect to the electron drift for the filling factors 1 and 1/3. Our analysis indicates that a neutral collective mode at the interaction-reconstructed edge is a proper candidate for the experimentally observed effect.

Phonon-mediated coupling of InGaAs/GaAs quantum-dot excitons to photonic crystal cavities.

We demonstrate that the emission characteristics of site-controlled InGaAs/GaAs single quantum dots embedded in photonic crystal slab cavities correspond to single confined excitons coupled to cavity modes, unlike previous reports of similar systems based on self-assembled quantum dots. By using polarization-resolved photoluminescence spectroscopy at different temperatures and a theoretical model, we show that the exciton-cavity interaction range is limited to the phonon sidebands. Photon-correlation and pump-power dependence experiments under nonresonant excitation conditions further establish that the cavity is fed only by a single exciton.

Sub-cycle switch-on of ultrastrong light-matter interaction.

Controlling the way light interacts with material excitations is at the heart of cavity quantum electrodynamics (QED). In the strong-coupling regime, quantum emitters in a microresonator absorb and spontaneously re-emit a photon many times before dissipation becomes effective, giving rise to mixed light-matter eigenmodes. Recent experiments in semiconductor microcavities reached a new limit of ultrastrong coupling, where photon exchange occurs on timescales comparable to the oscillation period of light. In this limit, ultrafast modulation of the coupling strength has been suggested to lead to unconventional QED phenomena. Although sophisticated light-matter coupling has been achieved in all three spatial dimensions, control in the fourth dimension, time, is little developed. Here we use a quantum-well waveguide structure to optically tune light-matter interaction from weak to ultrastrong and turn on maximum coupling within less than one cycle of light. In this regime, a class of extremely non-adiabatic phenomena becomes observable. In particular, we directly monitor how a coherent photon population converts to cavity polaritons during abrupt switching. This system forms a promising laboratory in which to study novel sub-cycle QED effects and represents an efficient room-temperature switching device operating at unprecedented speed.

Filling factor dependence of the fractional quantum Hall effect gap.

We directly measure the chemical potential jump in the low-temperature limit when the filling factor traverses the nu=1/3 and nu=2/5 fractional gaps in two-dimensional (2D) electron system in GaAs/AlGaAs single heterojunctions. In high magnetic fields B, both gaps are linear functions of B with slopes proportional to the inverse fraction denominator, 1/q. The fractional gaps close partially when the Fermi level lies outside. An empirical analysis indicates that the chemical potential jump for an ideal 2D electron system, in the highest accessible magnetic fields, is proportional to q(-1) B(1/2).

Direct measurements of fractional quantum Hall effect gaps.

We measure the chemical potential jump across the fractional gap in the low-temperature limit in the two-dimensional electron system of GaAs/AlGaAs single heterojunctions. In the fully spin-polarized regime, the gap for filling factor nu=1/3 increases linearly with the magnetic field and is coincident with that for nu=2/3, reflecting the electron-hole symmetry in the spin-split Landau level. In low magnetic fields, at the ground-state spin transition for nu=2/3, a correlated behavior of the nu=1/3 and nu=2/3 gaps is observed.

Morphology and composition of InAs/GaAs quantum dots.

Surface compositional maps of self-organized InAs/GaAs quantum dots were obtained with laterally resolved photoemission spectroscopy. We found a surface In concentration of about 0.85 at the center of the islands which decreases to 0.75 on the wetting layer. Comparison with concentration values found in the core of similar dots suggests a strong In segregation on the topmost surface layers of the dots and on the surrounding wetting layer. Furthermore, the morphological properties of the dots such as size and density have been measured with plan-view transmission electron microscopy and low energy electron microscopy.

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Role of charged residues in the catalytic mechanism of hepatitis C virus NS3 protease: electrostatic precollision guidance and transition-state stabilization.

Maturational cleavage of the hepatitis C virus polyprotein involves the viral chymotrypsin-like serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a highly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Using electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the active site led to changes in k(cat) values of a peptide substrate indicating that basic amino acids play a role in the stabilization of the transition state. Charge neutralization in the S5/S6 region increased the K(m) values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K(i) values of hexapeptide acids spanning P6-P1 (product inhibitors) were affected by charge neutralization in both the active site region and the S5/S6 region. Pre-steady-state kinetic data showed that the electrostatic surface potential is used by this enzyme to enhance collision rates between peptidic ligands and the active site. Calculations of the interaction energies of protease-substrate or protease-inhibitor complexes showed that electrostatic interaction energies oppose the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local electrostatic potentials.

Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its NS4A cofactor peptide.

The interaction of the hepatitis C virus (HCV) NS3 protease domain with its NS4A cofactor peptide (Pep4AK) was investigated at equilibrium and at pre-steady state under different physicochemical conditions. Equilibrium dissociation constants of the NS3-Pep4AK complex varied by several orders of magnitude depending on buffer additives. Glycerol, NaCl, detergents, and peptide substrates were found to stabilize this interaction. The extent of glycerol-induced stabilization varied in an HCV strain-dependent way with at least one determinant mapping to an NS3-NS4A interaction site. Conformational transitions affecting at least the first 18 amino acids of NS3 were the main energy barriers for both the association and the dissociation reactions of the complex. However, deletion of this N-terminal portion of the protease molecule only slightly influenced equilibrium dissociation constants determined under different physicochemical conditions. Limited proteolysis experiments coupled with mass spectrometric identification of cleavage fragments suggested a high degree of conformational flexibility affecting at least the first 21 residues of NS3. The accessibility of this region of the protease to limited chymotryptic digestion did not significantly change in any condition tested, whereas a significant reduction of chymotryptic cleavages within the NS3 core was detected under conditions of high NS3-Pep4AK complex affinity. We conclude the following: (1) The N-terminus of the NS3 protease that, according to the X-ray crystal structure, makes extensive contacts with the cofactor peptide is highly flexible in solution and contributes only marginally to the thermodynamic stability of the complex. (2) Affinity enhancement is accomplished by several factors through a general stabilization of the fold of the NS3 molecule.

Product inhibition of the hepatitis C virus NS3 protease.

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.

Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires.

Given the extent of hepatitis C virus (HCV) infection as a worldwide health problem and the lack of effective treatment, the development of anti-HCV drugs is an important and pressing objective. Previous studies have indicated that proteolytic events mediated by the NS3 protease of HCV are fundamental to the generation of an active viral replication apparatus, as unequivocably demonstrated for flaviviruses. As a result, the NS3 protease has become a major target for discovering anti-HCV drugs. To gain further insight into the biochemical and biophysical properties of the NS3 enzyme binding pocket(s) and to generate biological tools for developing antiviral strategies, we decided to engineer macromolecular ligands of the NS3 protease domain. Phage-displayed repertoires of minibodies ("minimized" antibody-like proteins) and human pancreatic secretory trypsin inhibitor were sampled by using the recombinant NS3 protease domain as a ligate molecule. Two protease inhibitors were identified and characterized biochemically. These inhibitors show marked specificity for the viral protease and potency in the micromolar range but display different mechanisms of inhibition. The implications for prospective development of low-molecular-weight inhibitors of this enzyme are discussed.

Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide.

The NS3 protein of the hepatitis C virus contains a serine protease that, upon binding to its cofactor, NS4A, is responsible for maturational cleavages that occur in the nonstructural region of the viral polyprotein. We have studied in vitro complex formation between the NS3 protease domain expressed in Escherichia coli and a synthetic peptide spanning the minimal domain of the NS4A cofactor. Complex dissociation constants in the low micromolar range were measured using different techniques such as activity titration, fluorescence titration, and pre-equilibrium analysis of complex formation. Cofactor binding was strictly dependent on the glycerol content of buffer solutions and was not significantly influenced by substrate saturation of the enzyme. NS4A peptide binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhancement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not differ significantly from that of the NS3-cofactor complex. Pre-equilibrium measurements of complex formation showed very low values for k(on), suggesting conformational transitions to be rate limiting for the association reaction.

Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease.

The HCV genome encodes, within the NS3 gene, a serine protease whose activity specifically cleaves the viral polyprotein precursor. Proteolytic processing of HCV polyprotein precursor by the viral NS3 proteinase is essential for virion maturation and designing specific inhibitors of this protease as possible anti-viral agents is a desirable and practical objective. With a view to studying both the function of HCV NS3 protease and to designing inhibitors of this enzyme, we directed our interest towards engineering macromolecular inhibitors of the viral protease catalytic activity. We describe here the affinity-selection and biochemical characterization of one inhibitor, cV(H)E2, a 'camelized' variable domain antibody fragment, isolated from a phage displayed synthetic repertoire, which is a potent and selective inhibitor of proteolysis by the NS3 enzyme. In addition to being useful as a biological probe to study the function of HCV protease, this inhibitor can serve as a potential pharmacophore model to design antivirals. Moreover, the results suggest a way of engineering improved human-derived small recognition units tailored for enzyme inhibition.