The studies in this thesis are mainly focused on the effects that the chaperonin mechanisms have on a bound target protein. Earlier studies have shown that the bacterial chaperonin GroEL plays an active role in unfolding a target protein during the initial binding. Here, the effects of the eukaryotic chaperonin TRiC’s mechanical action on a bound target protein were studied by fluorescence resonance energy transfer (FRET) measurements by attaching the fluorophore fluorescein to specific positions in the structure of the target protein, β-actin. Actin is an abundant eukaryotic protein and is dependent on TRiC to reach its native state. It was found that at the initial binding to TRiC, the actin structure is stretched, particularly across the nucleotide-binding site. This finding led to the conclusion that the binding-induced unfolding mechanism is conserved through evolution. Further studies indicated that in a subsequent step of the chaperonin cycle, the actin molecule collapses. This collapse leads to rearrangements of the structure at the nucleotide-binding cleft, which is also narrowed as a consequence. As a comparison to the productive folding of actin in the TRiC chaperonin system, FRET studies were also performed on actin interacting with GroEL. This is a non-productive interaction in terms of guiding actin to its native state. The study presents data indicating that the nucleotide-binding cleft in actin is not rearranged by GroEL in the same way as it is rearranged during the TRiC interaction. Thus, it could be concluded that although the general unfolding mechanism is conserved through the evolution of the chaperonins, an additional and specific binding to distinct parts of the actin molecule has evolved in TRiC. This specific binding leads to a directed unfolding and rearrangement of the nucleotide-binding cleft, which is vital for actin to reach its native state. The differences in the chemical properties of the actin-GroEL and the actin-TRiC complexes were also determined by measurements of fluorescein anisotropies and AEDANS emission shifts for probes attached to positions spread throughout the actin structure. The evolutionary aspects of the chaperonin mechanisms and the target protein binding were further investigated in another study. In this study, the prokaryotic homologue to actin, MreB, was shown to bind to both TRiC and GroEL. MreB was also shown to bind to the co-chaperonin GroES. In a separate study, the interaction between actin and the chaperone prefoldin was investigated. In vivo prefoldin interacts with non-native actin and transfers it to TRiC for subsequent and proper folding. In this homo-FRET study, it was shown that actin binds to prefoldin in a stretched conformation, similar to the initial binding of actin to TRiC. / On the day of the defence date the satus of article I was: In press.

In this thesis, I describe experiments that were designed to provide a better understanding of polymer diffusion during latex film formation. This step leads to the improvement of film mechanical properties. Polymer diffusion in these films was monitored by fluorescence resonance energy transfer. Current paint formulations contain Volatile Organic Compounds (VOCs) as plasticizers to facilitate polymer diffusion. The drawback of this technology is the release of VOCs to the atmosphere. VOCs are deleterious to the environment and contribute to smog and ground level ozone formation. The propensity of water, an indispensible part of any latex dispersion, to promote polymer diffusion was studied. Copolymers of poly (butyl acrylate-co-methyl methacrylate) and poly(ethylhexyl acrylate-co-tertiary butyl methacrylate) with similar glass transition temperatures but different hydrophobicity were compared. Polymer diffusion was monitored for films aged at different relative humidities. Water absorbed by the hydrophobic copolymer film was less efficient in promoting polymer diffusion than in the hydrophilic polymer. Only the fraction of water which is molecularly dissolved in the film participate actively in plasticization. Although water has low solubility in most latex polymers, molecularly dissolved water is more efficient than many traditional plasticizers. The possibility of modifying film formation behavior of acrylic dispersions with oligomers was studied by synthesizing hybrid polymer particles consisting of a high molecular weigh (high-M) polymer and an oligomer with the same composition. Oligomers with lower molecular weight are more efficient as diffusion promoters and have less deleterious effect on high-M polymer viscosity. A different set of hybrid particles were prepared in which the oligomer contained methacrylic acid units. The composition of the oligomer was tuned to be miscible with the high-M polymer when the acid groups were protonated but to phase separate when the acid groups were deprotonated. At basic pH, these particles adopt a core-shell morphology, with a shell rich in neutralized oligomers. After film formation, the oligomer shell retarded polymer diffusion. This retardation is expected to expand the time window during which the paint surface can be altered without leaving brush marks (open time). Short open time is a pressing problem in current technology.

In this thesis, I describe experiments that were designed to provide a better understanding of polymer diffusion during latex film formation. This step leads to the improvement of film mechanical properties. Polymer diffusion in these films was monitored by fluorescence resonance energy transfer. Current paint formulations contain Volatile Organic Compounds (VOCs) as plasticizers to facilitate polymer diffusion. The drawback of this technology is the release of VOCs to the atmosphere. VOCs are deleterious to the environment and contribute to smog and ground level ozone formation. The propensity of water, an indispensible part of any latex dispersion, to promote polymer diffusion was studied. Copolymers of poly (butyl acrylate-co-methyl methacrylate) and poly(ethylhexyl acrylate-co-tertiary butyl methacrylate) with similar glass transition temperatures but different hydrophobicity were compared. Polymer diffusion was monitored for films aged at different relative humidities. Water absorbed by the hydrophobic copolymer film was less efficient in promoting polymer diffusion than in the hydrophilic polymer. Only the fraction of water which is molecularly dissolved in the film participate actively in plasticization. Although water has low solubility in most latex polymers, molecularly dissolved water is more efficient than many traditional plasticizers. The possibility of modifying film formation behavior of acrylic dispersions with oligomers was studied by synthesizing hybrid polymer particles consisting of a high molecular weigh (high-M) polymer and an oligomer with the same composition. Oligomers with lower molecular weight are more efficient as diffusion promoters and have less deleterious effect on high-M polymer viscosity. A different set of hybrid particles were prepared in which the oligomer contained methacrylic acid units. The composition of the oligomer was tuned to be miscible with the high-M polymer when the acid groups were protonated but to phase separate when the acid groups were deprotonated. At basic pH, these particles adopt a core-shell morphology, with a shell rich in neutralized oligomers. After film formation, the oligomer shell retarded polymer diffusion. This retardation is expected to expand the time window during which the paint surface can be altered without leaving brush marks (open time). Short open time is a pressing problem in current technology.

Single-molecule spectroscopy has provided a means to uncover pathways and heterogeneities that were previously hidden beneath the ensemble average. Such heterogeneity, however, is often obscured by the artifacts of experimental noise and the occurrence of undesired processes within the experimental medium. This has subsequently caused in the need for new analytical methodologies. It is particularly important that objectivity be maintained in the development of new analytical methodology so that bias is not introduced and the results improperly characterized. The research presented herein identifies two such sources of experimental uncertainty, and constructs objective approaches to reduce their effects in the experimental results. The first, photoblinking, arises from the occupation of dark electronic states within the probe molecule, resulting in experimental data that is distorted by its contribution. A method based in Bayesian inference is developed, and is found to nearly eliminate photoblinks from the experimental data while minimally affecting the remaining data and maintaining objectivity. The second source of uncertainty is electronic shot-noise, which arises as a result of Poissonian photon collection. A method based in wavelet decomposition is constructed and applied to simulated and experimental data. It is iii found that, while making only one assumption, that photon collection is indeed a Poisson process, up to 75% of the shot-noise contribution may be removed from the experimental signal by the wavelet-based procedure. Lastly, in an effort to connect model-based approaches such as molecular dynamics simulation to model-free approaches that rely solely on the experimental data, a coarse-grained molecular model of a molecular ionic fluorophore diffusing within an electrostatically charged polymer brush is constructed and characterized. It is found that, while the characteristics of the coarse-grained simulation compare well with atomistic simulations, the model is lacking in its representation of the electrostatically-driven behavior of the experimental system.

Eukaryotic genomes are packaged as chromatin, which restricts access to the DNA by critical processes such as DNA replication, repair, and transcription. As a result, eukaryotic cells rely on ATP-dependent chromatin remodeling enzymes (remodelers) to alter the position, structure, and composition of nucleosomes. Understanding the mechanism and regulation of remodeling requires detailed information about transient intermediates of the remodeling process--a challenge ideally suited for single-molecule approaches. In particular, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure nanometer-scale distance changes between strategically placed donor and acceptor dyes to monitor nucleosome translocation in real-time. The mechanism(s) by which remodelers use the free energy of ATP hydrolysis to disrupt histone-DNA contacts and reposition nucleosomes are not well understood. Using smFRET, we show that remodeling by ISWI enzymes begins with a 7 base-pair (bp) step followed by subsequent 3 bp steps toward the exit-side of the nucleosome. These multi-bp steps are actually compound steps composed of 1 bp elementary steps. We discover that DNA movement on the entry side lags behind exit side translocation, which is contrary to previously proposed models. Based on our results, we propose a new integrated mechanism for nucleosome translocation by ISWI enzymes. In the physiological context, remodelers are highly regulated. We study the regulation of human ACF, a prototypical ISWI complex, by critical features of the nucleosomal substrate. First, we dissect how the nucleosome translocation cycle is affected by the linker DNA length and histone H4 tail. Next, we introduce mutations/deletions into conserved enzyme domains to determine the mechanism by which linker length information sensed by the Acfl accessory subunit is allosterically transmitted to the Snf2h catalytic subunit. Interestingly, we find that Acfl modulates the activity of Snf2h indirectly by interacting with the H4 tail in a linker-length dependent fashion. While the majority of our experiments focus on observing changes in nucleosome position, we also develop strategies for site-specific labeling of ISWI enzymes and demonstrate their use in the study of dynamic enzyme-substrate interactions and enzyme conformational changes.

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

Electronic Excitation Energy Transfer is an important intermolecular photophysical process that can affect the excited state lifetime of a chromophore. A molecule in an electronically excited state can return to the ground state by radiative as well as non-radiative processes. During the excited state lifetime, if the chromophore (energy donor) finds a suitable species (energy acceptor) nearby with resonant energy levels, it can transfer the excitation energy to that species and return to the ground state. This process is called Electronic Excitation Energy Transfer. When the energy donor is fluorescent, the process is called Fluorescence Resonance Energy Transfer (FRET) [1]. FRET is a non-radiative process that affects the fluorescence intensity as well as the excited state lifetime of the donor. It occurs due to the electrostatic coulombic interaction between the transition charge densities of the donor and the acceptor. The rate of energy transfer can be evaluated using the Fermi golden rule of quantum mechanics [2]. When the donor and the acceptor are separated by distances that are much larger in comparison with the sizes of the donor and the acceptor, the interaction between them can be thought of as that between their transition dipoles. In such a case, the interaction between the donor and the acceptor is dipolar and the rate of energy transfer has an R−6 dependence, where R is the distance between the donor and the acceptor [3]. This dependence has first been suggested theoretically by Forster in 1947 [4] followed by the experimental verification by Stryer and Haugland [5]. Since then the process has been used as a spectroscopic ruler to study the conformational dynamics of biopolymers like DNA, RNA, proteins etc [6]. A variety of dye molecules have been explored for donors and acceptors in FRET and the range of distances that can be measured using FRET involving dyes is in the range 1 − 10 nm. When the distances between the donor and the acceptor are not much larger in comparison with their sizes, the dipolar approximation to the interaction is not a very good approximation, thereby leading to deviations from the traditional R-6 dependence. Such non-R-6dependencies are found for polymers, quantum wells, quantum wires etc [7–9]. The interest in such dependencies is due to the need for developing nanoscopic rulers that can measure distances well beyond 10 nm. The objective of our work has been to study energy transfer from fluorophores to various kinds of acceptors that have extended charge densities and understand the distance dependence of the rate of energy transfer [10]. We use the Fermi golden rule as the starting point and develop analytical models for evaluating the rate as a function of the distance between the donor and the acceptor. We study the process of energy transfer from fluorescent dye molecules that serve as energy donors to a variety of energy acceptors namely, graphene, doped graphene, single-walled carbon nanotubes and metal nanoparticles. We also study transfer from fluorophores to a semiconducting sheet and a semiconducting tube of electronic charge density. There have been experimental studies in the literature of the fluorescence quenching of dyes near single-walled carbon nanotubes [11–13]. But, there are no studies of the distance dependence of rate. Single-walled carbon nanotubes can be thought of as rolled up sheets of graphene. However, interestingly, there were no reports of fluorescence quenching by graphene at the time when we thought of this possibility. Therefore, we first study the process of energy transfer from a fluorophore, which is kept at a distance z above a layer of graphene to the electronic energy levels of graphene. We find that the long range behavior of the rate has an z -4 dependence on the distance [14, 15]. From our study of transfer from pyrene to graphene, we find that fluorescence quenching can be experimentally observed up to a distance of ~ 30 nm, which is quite large in comparison with the traditional FRET limit (10 nm). Recent experiments that have been performed after our theory was reported have in fact observed the fluorescence quenching of dyes near graphene. Further, the process has been found to be very useful in fabricating devices based on graphene [16], in eliminating fluorescence signals in resonance Raman spectroscopy [17] and in visualizing graphene based sheets using fluorescence quenching microscopy [18]. The process has also been found to be useful in quantitative DNA analysis [19, 20]. We study the transfer of an amount of energy hΩ from a dye molecule to doped graphene [21]. We consider the shift of the Fermi level from the K-point into the conduction band of graphene as a result of doping and evaluate the rate of transfer. We find a crossover of the distance dependence of the rate from z -4 to exponential as the Fermi level is increasingly shifted into the conduction band, with the crossover occurring at a shift of the Fermi level by an amount hΩ/2. We study the process of transfer of excitation energy from a fluorophore kept at a distance d away from the surface of a carbon nanotube to the electronic energy levels of the nanotube. We find both exponential and d−5 behavior of the rate [22]. For the case of metallic nanotubes, when the emission energy of the fluorophore is less than a threshold, the dependence is exponential. Otherwise, it is d−5 . For the case of semiconducting nanotubes, we find that the rate follows an exponential dependence if the amount of energy that is transferred can cause only the excitonic transition of the tube. However, if any other band gap transition is allowed, the rate follows a d−5 dependence. For the case of transfer from pyrene to a (6, 4) nanotube, we find that energy transfer is appreciable up to a distance of ~ 17 nm. We then study the process of energy transfer from a fluorophore to a semiconducting sheet of electronic charge density [10]. We find that the rate has an z-4 dependence. For the case of transfer to a semiconducting tube, we find that the rate has a d -5dependence. The dependencies are in agreement with those obtained for graphene and carbon nanotubes respectively. This shows that the asymptotic distance dependencies are a consequence of the dimensionality of the transition charge densities and are robust. Strouse et al. [23, 24] have studied the process of energy transfer from the dye fluorescein to a 1.4 nm diameter gold nanoparticle. Double-stranded DNA molecules of various lengths were used to fix the distances between the donor and the acceptor. The rate was found to have a d-4distance dependence. They refer to this process as Nanoparticle Surface Energy Transfer (NSET) and the range of distances that can be measured using NSET is more than double that of the traditional FRET experiments. However, theoretical studies that consider the transfer to the plasmonic modes of the nanoparticle find a predominant R-6 dependence [25]. We study the process of energy transfer from the dye fluorescein to a 1.4 nm diameter gold nanoparticle considering the excitation of plasmons as well as electron-hole pairs of the nanoparticle [26]. We find that the rate follows the usual Forster type R−6 distance dependence at large distances. But, at short distances, there are contributions of the form R−-n with n > 6. This is due to the quadrupolar and octupolar modes of excitation of the nanoparticle, the rates corresponding to which have R-8 and R−-10 dependencies respectively. Recent calculations using DFT also find similar deviations at short distances [27].

No description available.

No / We describe a series of experiments designed to investigate the conformational transition that highly-branched polymers with ligands undergo when interacting with bacteria, a process that may provide a new sensing mechanism for bacterial detection. Fluorescent highly-branched poly(N-isopropyl acrylamide)s (HB-PNIPAM) were prepared by sequential self-condensing radical copolymerizations, using anthrylmethyl methacrylate (AMMA) and fluorescein-O-acrylate (FA) as fluorescent comonomers and 4-vinylbenzyl pyrrole carbodithioate as a branch forming monomer. Differences in reactivity necessitated to first copolymerize AMMA then react with FA in a separate sequential monomer feed step. Modifications of the chain ends produced vancomycin-functional derivatives (HB-PNIPAM-Van). The AMMA and FA labels allow probing of the conformational behaviour of the polymers in solution via Forster resonance energy transfer experiments. It was shown that interaction of this polymer's end groups with Staphylococcus aureus induced a macromolecular collapse. The data thus provide conclusive evidence for a conformational transition that is driven by binding to a bacterium.

Nanotechnology builds devices from the bottom up with atomic accuracy. Among the basic nano-components to fabricate such devices, semiconductor nanoparticle quantum dots (QDs), metal nanocrystals, proteins, and nucleic acids have attracted most interests due to their potential in optical, biomedical, and electronic areas. The major objective of this research was to prepare nano-components in order to fabricate functional nano-scale devices. This research consisted of three projects. In the first two projects, we incorporated two desirable characteristics of QDs, which are their abilities to serve as donors in fluorescence energy transfer (FRET) and surface energy transfer (SET) as well as to do multiplexing, to engineer QD-based nanoconjugates for optical and biomedical applications. Immobilizing luminescent semiconductor CdSe/ZnS QDs to a solid platform for QD-based biosensors offers advantages over traditional solution-based assays. In the first project, we designed highly sensitive CdSe/ZnS QD SET-based probes using gold nanoparticles (AuNPs) as FRET acceptors on polystyrene (PS) microsphere surfaces. The emission of PS-QD was significantly quenched and restored when the AuNPs were attached to and then removed from the surface. The probes were sensitive enough to analyze signals from a single bead and for use in optical applications. The new PS-QD-AuNP SET platform opens possibilities to carry out both SET and FRET assays in microparticle-based platforms and in microarrays. In the second project, we applied the QD-encoded microspheres in FRET-based analysis for bio-applications. QDs and Alexa Fluor 660 (A660) fluorophores are used as donors and acceptors respectively via a hairpin single stranded DNA. FRET between QD and A660 on the surface of polystyrene microspheres resulted in quenching of QD luminescence and increased A660 emission. QD emission on polystyrene x microspheres was restored when the targeted complementary DNA hybridized the hairpin strand and displaced A660 away from QDs. The third project involved fabrication of different nanoconjugates via self-assembly of template-based metal nanowires and metal nanoparticles using oligonucleotides as linkers. These nanoconjugates can serve as building blocks in nano-electronic circuits. The template method restricted the oligonucleotides attachment to the tip of the nanowires. Nanowires tagged with hybridizable DNA could connect to complementary DNA-modified metal crystals in a position-specific manner.