Luminescence from Droplet-Etched GaAs Quantum Dots at and Close to Room Temperature.

Epitaxially grown quantum dots (QDs) are established as quantum emitters for quantum information technology, but their operation under ambient conditions remains a challenge. Therefore, we study photoluminescence (PL) emission at and close to room temperature from self-assembled strain-free GaAs quantum dots (QDs) in refilled AlGaAs nanoholes on (001)GaAs substrate. Two major obstacles for room temperature operation are observed. The first is a strong radiative background from the GaAs substrate and the second a significant loss of intensity by more than four orders of magnitude between liquid helium and room temperature. We discuss results obtained on three different sample designs and two excitation wavelengths. The PL measurements are performed at room temperature and at T = 200 K, which is obtained using an inexpensive thermoelectric cooler. An optimized sample with an AlGaAs barrier layer thicker than the penetration depth of the exciting green laser light (532 nm) demonstrates clear QD peaks already at room temperature. Samples with thin AlGaAs layers show room temperature emission from the QDs when a blue laser (405 nm) with a reduced optical penetration depth is used for excitation. A model and a fit to the experimental behavior identify dissociation of excitons in the barrier below T = 100 K and thermal escape of excitons from QDs above T = 160 K as the central processes causing PL-intensity loss.

Single-molecule force spectroscopy of G-protein-coupled receptors.

The applicability of single-molecule force spectroscopy (SMFS) to characterize membrane proteins in vitro is developing rapidly and opening a wide range of fascinating possibilities to study how intra- and intermolecular interactions determine their structural stability and functional state. In particular, understanding how molecular interactions contribute to the functional state of G-protein-coupled receptors (GPCRs) is of importance because they mediate most of our physiological responses and act as therapeutic targets for a broad spectrum of diseases. In our review we focus on SMFS to characterize GPCRs embedded in their physiologically relevant membranes and exposed to physiologically relevant conditions. SMFS uses a molecularly sharp stylus to grasp the terminal end of a GPCR and to quickly unfold the receptor while recording interaction forces. The positional accuracy of SMFS localizes these interactions to structural segments of the GPCR whereas the sensitivity of SMFS enables their stabilizing interaction forces to be quantified. To further investigate the kinetic, energetic and mechanical properties of the structural segments, dynamic SMFS (DFS) probes their stability over a wide range of loading rates. These parameters provide insight into the energy landscape that provides information on the structural and functional properties of the GPCRs. Selected highlights exemplify the application of SMFS to characterize inter- and intramolecular interactions, which change the properties of GPCRs in relation to their functional state (e.g., ligand binding), diseased state (e.g., mutation), or lipid environment such as cholesterol.

Cholesterol increases kinetic, energetic, and mechanical stability of the human ß2-adrenergic receptor.

The steroid cholesterol is an essential component of eukaryotic membranes, and it functionally modulates membrane proteins, including G protein-coupled receptors. To reveal insight into how cholesterol modulates G protein-coupled receptors, we have used dynamic single-molecule force spectroscopy to quantify the mechanical strength and flexibility, conformational variability, and kinetic and energetic stability of structural segments stabilizing the human ß(2)-adrenergic receptor (ß(2)AR) in the absence and presence of the cholesterol analog cholesteryl hemisuccinate (CHS). CHS considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and functional relationship of ß(2)AR. One exception was the structural core segment of ß(2)AR, which establishes multiple ligand binding sites, and its properties were not significantly influenced by CHS.

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase.

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.

Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human ß2 adrenergic receptor.

G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human ß2 adrenergic receptor (ß2 AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of ß2 AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.

Single-molecule force spectroscopy from nanodiscs: an assay to quantify folding, stability, and interactions of native membrane proteins.

Single-molecule force spectroscopy (SMFS) can quantify and localize inter- and intramolecular interactions that determine the folding, stability, and functional state of membrane proteins. To conduct SMFS the membranes embedding the membrane proteins must be imaged and localized in a rather time-consuming manner. Toward simplifying the investigation of membrane proteins by SMFS, we reconstituted the light-driven proton pump bacteriorhodopsin into lipid nanodiscs. The advantage of using nanodiscs is that membrane proteins can be handled like water-soluble proteins and characterized with similar ease. SMFS characterization of bacteriorhodopsin in native purple membranes and in nanodiscs reveals no significant alterations of structure, function, unfolding intermediates, and strengths of inter- and intramolecular interactions. This demonstrates that lipid nanodiscs provide a unique approach for in vitro studies of native membrane proteins using SMFS and open an avenue to characterize membrane proteins by a wide variety of SMFS approaches that have been established on water-soluble proteins.

Masking of transmembrane-based retention signals controls ER export of gamma-secretase.

gamma-Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of gamma-secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER-retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of gamma-secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER-retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER-retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent gamma-secretase complex could mask TMD-based ER-retention/retrieval signals to allow plasma membrane transport of fully assembled gamma-secretase.