The (Un)Folding of Multidomain Proteins Through the Lens of Single-molecule Force-spectroscopy and Computer Simulation

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Synthesis, structure and magnetic properties of heterometallic complexes towards single-molecule magnets using flexible aminopolyol ligands

No abstract available.

Performance evaluation of DCA and SRC on a single bot detection

Malicious users try to compromise systems using new techniques. One of the recent techniques used by the attacker is to perform complex distributed attacks such as denial of service and to obtain sensitive data such as password information. These compromised machines are said to be infected with malicious software termed a “bot”. In this paper, we investigate the correlation of behavioural attributes such as keylogging and packet flooding behaviour to detect the existence of a single bot on a compromised machine by applying (1) Spearman’s rank correlation (SRC) algorithm and (2) the Dendritic Cell Algorithm (DCA). We also compare the output results generated from these two methods to the detection of a single bot. The results show that the DCA has a better performance in detecting malicious activities.

Solid state micro and nanopore sensors for single entity detection

A general description of the work presented in this thesis can be divided into three areas of interest: micropore fabrication, nanopore modification, and their applications. The first part of the thesis is related to the novel, reliable, cost-effective, potable, mass-productive, robust, and ease of use micropore flowcell that works based on the RPS technique. Based on our first goal, which was finding an alternate materials and processes that would shorten production times while lowering costs and improving signal quality, the polyimide film was used as a substrate to create precise pores by femtosecond laser, and the resulting current blockades of different sizes of the nanoparticles were recorded. Based on the results, the device can detecting nano-sized particles by changing the current level. The experimental and theoretical investigation, scanning electron microscopy, and focus ion beam were performed to explain the micropore's performance. The second goal was design and fabrication of a leak-free, easy-to-assemble, and portable polymethyl methacrylate flowcell for nanopore experiments. Here, ion current rectification was studied in our nanodevice. We showed a self-assembly-based, controllable, and monitorable in situ Poly(l-lysine)- g-poly(ethylene glycol) coating method under voltage-driven electrolyte flow and electrostatic interaction between nanopore walls and PLL backbones. Using designed nanopore flowcell and in situ monolayer PLL-g-PEG functionalized 20±4 nm SiN nanopores, we observed non-sticky α-1 anti-trypsin protein translocation. additionally, we could show the enhancement of translocation events through this non-sticky nanopore, and also, estimate the volume of the translocated protein. In this study, by comparing the AAT protein translocation results from functionalized and non-functionalized nanopore we demonstrated the 105 times dwell time reduction (31-0.59ms), 25% amplitude enhancement (0.24-0.3 nA), and 15 times event’s number increase (1-15events/s) after functionalization in 1×PBS at physiological pH. Also, the AAT protein volume was measured, close to the calculated AAT protein hydrodynamic volume and previous reports.

Produção de substratos sers eficientes através da deposição de ouro sobre um molde de microesferas de poliestireno

This work reports on the SERS activity of a nanostructured substrate that was obtained by electrodepositing gold over a template consisting of polystyrene microspheres. This substrate displayed superior SERS performance for the detection of 4-merctaptopyridine as compared to a conventional roughened Au electrode. In order to investigate the substrate capability for the detection at low concentration limits, a series of Rhodamine 6G (1 nM) spectra were registered. Our spectral dynamics data is in agreement with single-molecule behavior, showing that the control over the substrate morphology is crucial to enable the production of highly reproducible and sensitive SERS substrates.

Produção de substratos sers eficientes através da deposição de ouro sobre um molde de microesferas de poliestireno

This work reports on the SERS activity of a nanostructured substrate that was obtained by electrodepositing gold over a template consisting of polystyrene microspheres. This substrate displayed superior SERS performance for the detection of 4-merctaptopyridine as compared to a conventional roughened Au electrode. In order to investigate the substrate capability for the detection at low concentration limits, a series of Rhodamine 6G (1 nM) spectra were registered. Our spectral dynamics data is in agreement with single-molecule behavior, showing that the control over the substrate morphology is crucial to enable the production of highly reproducible and sensitive SERS substrates.

Terahertz spectroscopy and imaging at the nanoscale for biological and security applications

The chemical specificity of terahertz spectroscopy, when combined with techniques for sub-wavelength sensing, is giving new understanding of processes occurring at the nanometre scale in biological systems and offers the potential for single molecule detection of chemical and biological agents and explosives. In addition, terahertz techniques are enabling the exploration of the fundamental behaviour of light when it interacts with nanoscale optical structures, and are being used to measure ultrafast carrier dynamics, transport and localisation in nanostructures. This chapter will explain how terahertz scale modelling can be used to explore the fundamental physics of nano-optics, it will discuss the terahertz spectroscopy of nanomaterials, terahertz near-field microscopy and other sub-wavelength techniques, and summarise recent developments in the terahertz spectroscopy and imaging of biological systems at the nanoscale. The potential of using these techniques for security applications will be considered.

SURFACE-ENHANCED RAMAN SCATTERING: METAL NANOSTRUCTURES COATED WITH LANGMUIR-BLODGETT FILMS

This review deals with surface-enhancved Raman scattering (SERS) employing Langmuir-Blodgett (LB) films, which serve as model systems for developing theoretical and experimental studies to elucidate the SERS effect. In addition, LB films have be used as integral parts of molecular architectures for SERS-active substrates. On the other hand, SERS and surface-enhaced resonant Raman scattering (SERRS) have allowed various properties of LB films to be investigated, especially those associated with molecular-level interactions. In the paper, emphasis is placed on single molecule detection (SMD), where the target molecule is diluted on an LB matrix of spectral silent material (low Raman cross section). The perspectives and challenges for combining SERS and LB films are also discussed.

Surface-enhanced raman scattering: metal nanostructures coated with langmuir-blodgett films

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Plasmonic nanoparticles as sensors : a novel approach to quantify adsorption and diffusion kinetics

Biosensors find wide application in clinical diagnostics, bioprocess control and environmental monitoring. They should not only show high specificity and reproducibility but also a high sensitivity and stability of the signal. Therefore, I introduce a novel sensor technology based on plasmonic nanoparticles which overcomes both of these limitations. Plasmonic nanoparticles exhibit strong absorption and scattering in the visible and near-infrared spectral range. The plasmon resonance, the collective coherent oscillation mode of the conduction band electrons against the positively charged ionic lattice, is sensitive to the local environment of the particle. I monitor these changes in the resonance wavelength by a new dark-field spectroscopy technique. Due to a strong light source and a highly sensitive detector a temporal resolution in the microsecond regime is possible in combination with a high spectral stability. This opens a window to investigate dynamics on the molecular level and to gain knowledge about fundamental biological processes.rnFirst, I investigate adsorption at the non-equilibrium as well as at the equilibrium state. I show the temporal evolution of single adsorption events of fibrinogen on the surface of the sensor on a millisecond timescale. Fibrinogen is a blood plasma protein with a unique shape that plays a central role in blood coagulation and is always involved in cell-biomaterial interactions. Further, I monitor equilibrium coverage fluctuations of sodium dodecyl sulfate and demonstrate a new approach to quantify the characteristic rate constants which is independent of mass transfer interference and long term drifts of the measured signal. This method has been investigated theoretically by Monte-Carlo simulations but so far there has been no sensor technology with a sufficient signal-to-noise ratio.rnSecond, I apply plasmonic nanoparticles as sensors for the determination of diffusion coefficients. Thereby, the sensing volume of a single, immobilized nanorod is used as detection volume. When a diffusing particle enters the detection volume a shift in the resonance wavelength is introduced. As no labeling of the analyte is necessary the hydrodynamic radius and thus the diffusion properties are not altered and can be studied in their natural form. In comparison to the conventional Fluorescence Correlation Spectroscopy technique a volume reduction by a factor of 5000-10000 is reached.

Specific binding of proinsulin C-peptide to human cell membranes

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand–membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with Kass > 3.3 × 109 M−1. Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 × 10−4 s−1. The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative d-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Confocal microscopy applied to the study of single entity fluorescence and light scattering

A sample scanning confocal optical microscope (SCOM) was designed and constructed in order to perform local measurements of fluorescence, light scattering and Raman scattering. This instrument allows to measure time resolved fluorescence, Raman scattering and light scattering from the same diffraction limited spot. Fluorescence from single molecules and light scattering from metallic nanoparticles can be studied. First, the electric field distribution in the focus of the SCOM was modelled. This enables the design of illumination modes for different purposes, such as the determination of the three-dimensional orientation of single chromophores. Second, a method for the calculation of the de-excitation rates of a chromophore was presented. This permits to compare different detection schemes and experimental geometries in order to optimize the collection of fluorescence photons. Both methods were combined to calculate the SCOM fluorescence signal of a chromophore in a general layered system. The fluorescence excitation and emission of single molecules through a thin gold film was investigated experimentally and modelled. It was demonstrated that, due to the mediation of surface plasmons, single molecule fluorescence near a thin gold film can be excited and detected with an epi-illumination scheme through the film. Single molecule fluorescence as close as 15nm to the gold film was studied in this manner. The fluorescence dynamics (fluorescence blinking and excited state lifetime) of single molecules was studied in the presence and in the absence of a nearby gold film in order to investigate the influence of the metal on the electronic transition rates. The trace-histogram and the autocorrelation methods for the analysis of single molecule fluorescence blinking were presented and compared via the analysis of Monte-Carlo simulated data. The nearby gold influences the total decay rate in agreement to theory. The gold presence produced no influence on the ISC rate from the excited state to the triplet but increased by a factor of 2 the transition rate from the triplet to the singlet ground state. The photoluminescence blinking of Zn0.42Cd0.58Se QDs on glass and ITO substrates was investigated experimentally as a function of the excitation power (P) and modelled via Monte-Carlo simulations. At low P, it was observed that the probability of a certain on- or off-time follows a negative power-law with exponent near to 1.6. As P increased, the on-time fraction reduced on both substrates whereas the off-times did not change. A weak residual memory effect between consecutive on-times and consecutive off-times was observed but not between an on-time and the adjacent off-time. All of this suggests the presence of two independent mechanisms governing the lifetimes of the on- and off-states. The simulated data showed Poisson-distributed off- and on-intensities, demonstrating that the observed non-Poissonian on-intensity distribution of the QDs is not a product of the underlying power-law probability and that the blinking of QDs occurs between a non-emitting off-state and a distribution of emitting on-states with different intensities. All the experimentally observed photo-induced effects could be accounted for by introducing a characteristic lifetime tPI of the on-state in the simulations. The QDs on glass presented a tPI proportional to P-1 suggesting the presence of a one-photon process. Light scattering images and spectra of colloidal and C-shaped gold nano-particles were acquired. The minimum size of a metallic scatterer detectable with the SCOM lies around 20 nm.

The role of AKT-mediated phosphorylation in suppression of MAD1 and the development of new approach in protein complex detection

MAX dimerization protein 1 (MAD1) is a basic-helix-loop-helix transcription factors that recruits transcription repressor such as HDAC to suppress target genes transcription. It antagonizes to MYC because the promoter binding sites for MYC are usually also serve as the binding sites for MAD1 so they compete for it. However, the mechanism of the switch between MYC and MAD1 in turning on and off of genes' transcription is obscure. In this study, we demonstrated that AKT-mediated MAD1 phosphorylation inhibits MAD1 transcription repression function. The association between MAD1 and its target genes' promoter is reduced after been phosphorylated by AKT; therefore, consequently, allows MYC to occupy the binding site and activates transcription. Mutation of such phosphorylation site abrogates the inhibition from AKT. In addition, functional assays demonstrated that AKT suppressed MAD1-mediated transcription repression of its target genes hTERT and ODC. Cell cycle and cell growth were also been released from inhibition by MAD1 in the presents of AKT. Taken together, our study suggests that MAD1 is a novel substrate of AKT and AKT-mediated MAD1 phosphorylation inhibits MAD1function; therefore, activates MAD1 target genes expression. ^ Furthermore, analysis of protein-protein interaction is indispensable for current molecular biology research, but multiplex protein dynamics in cells is too complicated to be analyzed by using existing biochemical methods. To overcome the disadvantage, we have developed a single molecule level detection system with nanofluidic chip. Single molecule was analyzed based on their fluorescent profile and their profiles were plotted into 2 dimensional time co-incident photon burst diagram (2DTP). From this 2DTP, protein complexes were characterized. These results demonstrate that the nanochannel protein detection system is a promising tool for future molecular biology. ^

Spin-detection in a quantum electromechanical shuttle system

We study the electrical transport of a harmonically bound, single-molecule C-60 shuttle operating in the Coulomb blockade regime, i.e. single electron shuttling. In particular, we examine the dependance of the tunnel current on an ultra-small stationary force exerted on the shuttle. As an example, we consider the force exerted on an endohedral N@C-60 by the magnetic field gradient generated by a nearby nanomagnet. We derive a Hamiltonian for the full shuttle system which includes the metallic contacts, the spatially dependent tunnel couplings to the shuttle, the electronic and motional degrees of freedom of the shuttle itself and a coupling of the shuttle's motion to a phonon bath. We analyse the resulting quantum master equation and find that, due to the exponential dependence of the tunnel probability on the shuttle-contact separation, the current is highly sensitive to very small forces. In particular, we predict that the spin state of the endohedral electrons of N@C-60 in a large magnetic gradient field can be distinguished from the resulting current signals within a few tens of nanoseconds. This effect could prove useful for the detection of the endohedral spin-state of individual paramagnetic molecules such as N@C-60 and P@C-60, or the detection of very small static forces acting on a C-60 shuttle.