Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.

Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson's disease, with an improved sensitivity of >100,000-fold over bulk measurements.

The authors would like to include the following authors and their contributions and funding details: Author list: Yann Gambin, Nichole Giles, Ailis O'Carroll , Mark E. Polinkovsky, Wayne Johnston, Dominic J.B Hunter, Kirill Alexandrov, Kate Schroder, Emma Sierecki. Affiliations: EMBL Australia Node in Single Molecule Science, University of New South Wales, Kensington, NSW 2052, Australia. The Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia. IMB Centre for Inflammation and Disease Research, University of Queensland, St Lucia, QLD 4072, Australia. Author's contributions: N.G., Y.G. and E.S. carried out the cell-free experiments. D.H. contributed the cell-free reagent. N.G., A.O.C., M.E.P., Y.G. and E.S. performed single-molecule coincidence analysis and AlphaScreen experiments and analyzed data. W.J. contributed to the development of the cell-free expression system. K.A. contributed to the development of the cell-free expression system and to formulation of the research objective. K.S. contributed to design and conceptualization of the study, and expertise in inflammasome biology. E.S. and Y.G. designed the study. Y.G., E.S., K.S. and N.G. drafted the manuscript. All authors read and approved the final manuscript. Funding: The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland. Y.G. and K.S. are supported by Australian Research Council Future Fellowships (FT110100478 to Y.G., FT130100361 to K.S.). The National Health and Medical Research Council supported the research (Program Grants 511005 and APP1037320 to K.A., and APP1100771 to Y.G. and E.S.). The authors would like to apologize for any inconvenience caused.

Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in "overdrive" by producing a single micron-sized "speck." By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the "prion-like" conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level.

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule "counting" methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.

Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.

ABSTRACTOver the last half century, a range of cell‐free protein expression systems based on pro‐ and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species‐independent translation initiation sequence to express and characterize 87 N‐terminally GFP‐tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania‐based (LTE) cell‐free systems. Using a combination of single‐molecule fluorescence spectroscopy, SDS‐PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full‐length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species—particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE‐produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa‐ and LTE‐produced proteins enable their analysis without purification and make them suitable for analysis of multi‐domain eukaryotic proteins. Biotechnol. Bioeng. 2016;113: 292–300. © 2015 Wiley Periodicals, Inc.

The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10–15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations. Temperature jump technique is widely used to probe the fast dynamics of protein and DNA folding, but constrained to modest temperature control. Here, the authors use a microfluidic device combined with an infrared laser to heat or cool DNA hairpins up to 70 °C on a microsecond time scale.

Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pair-wise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure.

The NLRP3 and NLRP12 inflammasome complexes are a subset of the Nod‐like receptor family, known to play a key role in the innate immune system. They act by activating caspase‐1 in response to pathogens, as well as endogenous and environmental stimuli. The NLRP3 inflammasome consists of the sensing protein, NLRP3, adaptor protein ASC, and pro‐caspase‐1. The proposed structure of the complex is based on the effector domains on the proteins, although the assembly of the active complex remains unclear. Even less is known about the NLRP12 inflammasome.We used a combination of Leishmania tarentolae cell‐free expression system, AlphaScreen, and single molecule fluorescence spectroscopy to investigate the assembly of NLRP3 and NLRP12 inflammasomes. Our results suggest that the NLRP3 and NLRP12 inflammasomes have different mechanisms of assembly. We observed NLRP3 and NLRP12 forming oligomers of distinct size. While NLRP3 can bind to ASC and pro‐caspase‐1 independently, association of NLRP12 with pro‐caspase‐1 requires ASC. Interestingly, we observed that aggregation of ASC is concentration dependent and nucleation of ASC has characteristics of the seeding model proposed for prions.Grant Funding Source: Australian Research Council

The Mediator Complex plays a major role in regulating gene transcription in eukaryotes. One of its fundamental roles is to integrate inputs from a variety of transcription factors to control the gene’s transcriptional state. The majority of these transcription factors are thought to bind to the Mediator’s Tail subdomain. In humans, the Tail is composed of eight subunit proteins, whose structure and arrangement remain unknown, largely due to difficulties in expressing and purifying them. Moreover, only a few interactions between transcription factors and some of the Tail subunits have been found.To overcome difficulties in protein expression and interaction analysis, we used the Leishmania cell‐free protein expression system and pair‐wise measurements of protein interactions via the AlphaScreen assay. These methods allowed us to build a model of the interactions between protein subunits in the Mediator Tail. As validation, we pulled down all the component proteins in the Tail subdomain from a single cell‐free co‐expression. The same techniques were used to test the binding of various transcription factors to the Tail subunits. Our results show that different families of transcription factors bind to distinct groups of subunits in the Tail. Combined with our model of the Tail, this data reveals binding pockets on the Tail’s surface.Grant Funding Source: Supported by Australian Research Council

Gene transcription is an exquisitely regulated phenomenon in which multiple layers of protein‐protein interactions participate in the integration of the signal. The mechanism is mediated through interaction of transcription factors with a large array of regulatory proteins. “Activators” and “repressors” binding DNA upstream of the transcription factors provide positive or negative signals. Proteins called “co‐activators” or “adaptors” that cannot bind DNA by themselves, are responsible for tethering the transcription factors to the RNA polymerase and coordinate the influence of activators and repressors. These adaptors often form large protein assemblies. The largest complex, called “Mediator” is comprised of up to 25 subunits that are grouped into four modules called head, middle, CDK and tail, with a total mass of approximately 1 MDa.To study this adaptor protein complex, a rapid access to numerous recombinant subunits is required. Co‐expression of the subunits is also key to succeed as the level of disorder in subunits of Mediator is far greater than in other multiprotein complexes of similar size. Here, for the first time, we present a model for the architecture of the entire Mediator complex, obtained using a novel combination of protein analysis techniques. All possible subunit pairs were co‐expressed in a Leishmania tarentolae‐based cell‐free expression system and screened for their ability to interact by an AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen). Our results correlate with the published structure of the Mediator head and reveal intricate interactions between the subunits.Grant Funding Source: Supported by the ARC and NHMRC

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

A protein's conformation depends on the protein's chemical and physical environment, including the temperature, pH, and denaturant concentration. The changes in protein structure can be visualized by Förster resonance energy transfer (FRET) between donor and acceptor fluorescent dyes bound to separate residues on the protein. One method of studying protein folding kinetics is the temperature jump, where a protein sample is quickly heated by a laser pulse to trigger a change in the protein conformation. During and after the pulse, a fluorescence excitation laser illuminates the protein sample and the FRET signal is collected. Traditional temperature jump methods have very short heating times (∼1 ns), facilitating the observation of protein folding events triggered by heating. However, due to the relatively large heated volume (∼1 nL), the long cooling time (hundreds of μs) obscures cooling-driven protein folding events (tens of μs). In order to observe fast folding events, the protein sample is often placed in non-native conditions: its unfolded state is imposed by low temperatures, usually at a high denaturant concentration, so that the heating laser pulse will induce the protein to refold. We have designed a novel system, which we call the inverse temperature jump or iT-jump, that enables observations of fast folding transitions by cooling the protein sample on a timescale of <1 μs. The rapid cooling is made possible by placing the sample in a shallow microfluidic channel and tightly focusing the heating laser beam to a volume of ∼1 fL. The proposed system inverts the existing T-jump paradigm, as the heating pulse unfolds the protein and the fast cooling transition allows it to refold. As a demonstration of our system's capabilities, we have studied the folding behavior of BBL, a small, fast-folding protein, under various denaturant conditions.

We present a system consisting of a microfluidic device made of gas-permeable polydimethylsiloxane (PDMS) with two layers of microchannels and a computer-controlled multi-channel gas mixer. Concentrations of oxygen in the liquid-filled flow channels of the device are imposed by flowing gas mixtures with desired oxygen concentrations through gas channels directly above the flow channels. Oxygen gradients with different linear, exponential, and non-monotonic shapes are generated in the same liquid-filled microchannel and reconfigured in real time. The system can be used to study directed migration of cells and the development of cell and tissue cultures under gradients of oxygen.

We describe the design, operation, and applications of two microfluidic devices that generate series of concentrations of oxygen, [O(2)], by on-chip gas mixing. Both devices are made of polydimethylsiloxane (PDMS) and have two layers of channels, the flow layer and the gas layer. By using in-situ measurements of [O(2)] with an oxygen-sensitive fluorescent dye, we show that gas diffusion through PDMS leads to equilibration of [O(2)] in an aqueous solution in the flow layer with [O(2)] in a gas injected into the gas layer on a time scale of approximately 1 sec. Injection of carbon dioxide into the gas layer causes the pH in the flow layer to drop within approximately 0.5 sec. Gas-mixing channel networks of both devices generate series of 9 gas mixtures with different [O(2)] from two gases fed to the inlets, thus creating regions with 9 different [O(2)] in the flow layer. The first device generates nitrogen-oxygen mixtures with [O(2)] varying linearly between 0 and 100%. The second device generates nitrogen-air mixtures with [O(2)] varying exponentially between 0 and 20.9%. The flow layers of the devices are designed for culturing bacteria in semi-permeable microchambers, and the second device is used to measure growth curves of E. coli colonies at 9 different [O(2)] in a single experiment. The cell division rates at [O(2)] of 0, 0.2, and 0.5% are found to be significantly different, further validating the capacity of the device to set [O(2)] in the flow layer with high precision and resolution. The degree of control of [O(2)] achieved in the devices and the robustness with respect to oxygen consumption due to respiration would be difficult to match in a traditional large-scale culture. The proposed devices and technology can be used in research on bacteria and yeast under microaerobic conditions and on mammalian cells under hypoxia.

The young field of microfluidics has been growing due to its utility in chemical and biological applications. Microfluidic devices can be rapidly and inexpensively fabricated from silicone elastomers, making them ideal for prototyping and subsequent production. Further, the behavior of fluid flows in micrometer-diameter channels can be accurately predicted - due to the properties of laminar flow and purely diffusive mixing - decreasing experimental uncertainties, while allowing access to a wide range of experiments impossible with traditional methods. The projects presented here fall into two separate areas of biophysics, although they are all facilitated by microfluidics. Chapter 2 deals with the control of the gas content in the medium of cell cultures. This is an important consideration, as the oxygen concentration, [O₂], available to cells has been shown to affect their metabolism, growth, and gene expression. The first project is a microfluidic chemostat supplying nine different [O₂] to bacteria growing in chambers beneath the gas channels. Here, we compared the growth rates of E. coli growing at nine different [O₂] simultaneously. Section 2.2 introduces a multi-channel, computer- controlled gas mixer that can provide up to ten arbitrary gas mixtures to a microfluidic device. Finally, Section 2.3 describes gas control strips for use with mammalian cell cultures in standard multiwell culture plates. These gas control strips allow cell culture media in different rows of wells to contain different [O₂]. Chapter 3 describes a novel system to rapidly heat and cool a small volume of solution of biological macromolecules using time -controlled deposition of heat into a small volume with a focused infrared laser beam. By fluorescently labeling the molecules, their conformational changes due to temperature shifts can be observed. This system improves the time resolution of the cooling transition over traditional methods by at least two orders of magnitude, down to one microsecond. Further, the temperature change from the laser heating pulse is several times larger than with other techniques. We used this system to measure the kinetics of fast DNA hairpin folding and unfolding under varying salt concentrations