No description available.

The analysis of protein residues recovered from archaeological artifacts provides a unique opportunity to reveal new information about past societies. However, many scientists are currently unwilling to accept protein-based results due to problems in method development and a basic lack of agreement regarding the ability of proteins to bind to, and preserve within, artifacts such as pottery. In this paper, I address these challenges by conducting a two-phase experiment. First, I quantitatively evaluate the tendency of proteins to sorb to ceramic matrices by using total organic carbon analysis and spectrophotometric assays to analyze samples of experimentally cooked ceramic. I then test a series of solvent and physical parameters in order to develop an optimized method for extracting and preparing protein residues for identification via mass spectrometry. Results demonstrate that protein strongly sorbs to ceramic and is not easily removed, despite repeated washing, unless an appropriate extraction strategy is used. This has implications for the future of paleodietary, conservation ecology and forensic research in that it suggests the potential for recovery of aged or even ancient proteins from ceramic matrices.

High-voltage activated calcium channels underlie many critical functions in excitable cells and their dysfunction has been implicated in a myriad of cardiovascular and neurological diseases. These channels are multimeric protein complexes composed of α1, β, and α2δ subunits; currently, all calcium channel blockers target either the pore-forming α1 or extracellular-facing α2δ auxiliary subunit. These pharmacological agents have been invaluable in delineating the individual function of each subunit within excitable cells that express multiple calcium channels. Yet, no current tool allows similar pharmacological dissection of individual cytosolic β subunits, preventing our understanding of how distinct β subunits affect the function of calcium channel complexes. Further, small-molecule calcium channel blockers are highly-valued therapeutics for certain conditions, yet their propensity for off-target effects precludes their use in other diseases. In certain applications, genetically-encoded calcium channel blockers may enable channel inhibition with greater tissue-precision and versatility than is achievable with small molecules. Previous work that found the family of RGK proteins powerfully inhibits high-voltage activated calcium channels in part via an association with the β subunit. However, the myriad functions of RGK proteins limit the utility of this approach. In this work, we circumvent this issue by isolating single-domain antibodies (nanobodies) that target the auxiliary CaVβ subunit. We then paired these nanobodies with the powerful enzymatic activity of the HECT domain E3 ubiquitin ligase Nedd4L, to selectively target the calcium channel for ubiquitination. We found this strategy effectively eliminated functional calcium channels from the surface of HEK293 cells, myocytes, and DRG neurons. This modular design permitted us to characterize a pan-β inhibitor (CaV-aβlator) in chapter 2 while refining the approach with a β1-selective channel inhibitor in chapter 3. In chapter 4 I demonstrate that it is possible to hijack the endogenous ubiquitin machinery of the cell by creating Divas: divalent nanobodies that are capable of recruiting endogenous Nedd4L to regulate the calcium channel. Finally, we demonstrate the potential for these genetically-encoded calcium inhibitors to be employed as therapeutic agents by targeting CaV-aβlator to sensory neurons in order to reduce the onset of neuropathic pain. Altogether, this work lays the foundation for nanobody-based genetically-encoded calcium channel inhibitors that have the potential to achieve superior precision in regards to molecular and tissue specificity.

Despite the variety of successful reports of the preservation, recovery, and identification of archaeological proteins in general, there are few positive reports regarding mass spectrometry-based identification of ceramic-bound proteins. In large part, this shortage is due to the lack of consideration for the unique taphonomic histories of such residues and, in general, methods development. Further, because negative results are rarely published, there is no baseline to which results can be compared. This paper attempts to address these challenges via a multi-pronged approach that uses mass spectrometry and complementary approaches to evaluate ceramic-bound protein preservation in both controlled, actualistic experiments, and in archaeological artifacts. By comparing the results obtained from protein-spiked, experimentally-aged ceramic to those obtained from both faunal and ceramic archaeological materials, an enhanced perspective on protein preservation and subsequent recovery and identification is revealed. This perspective, focusing on taphonomy, reveals why negative results may be the norm for ceramic artifacts when non-targeted methods are employed, and provides insight into how further method development may improve the likelihood of obtaining positive results.

Neutrophils and macrophages accumulate at sites of inflammation and cause chronic inflammation leading to various diseases. Therefore, to better understand chronic disease pathways it is important to investigate the properties of macrophage accumulation in inflamed tissues. The I-domain of the macrophage receptor integrin aDb2 plays a vital role in macrophage retention by binding to CEP (carboxyethyl pyrrole), a ligand available at inflammatory sites. This thesis mainly focuses on evaluating the binding site within integrin aDb2 that binds carboxyethyl pyrrole (CEP)-modified proteins. So, a recombinant plasmid construct containing the integrin I-domain was developed. Seven non-conserved amino acids were mutated by PCR-site-directed mutagenesis to create a mutant construct. After expressing in E. coli, the binding affinities of wild-type and mutant I-domains to CEP were analyzed using biolayer interferometry. It was found that a patch of seven positively charged amino acids contributes to the strong binding of the I domain to CEP.

No description available.

No description available.

Affinity molecules can serve as precision tools for selective recognition and measurement of specific biomolecules in the fields of therapeutic drug monitoring and quality control in recombinant protein production. In therapeutic drug monitoring, affinity molecules can enable the accurate quantification of drug concentrations within physiological fluids, enhancing both the safety and efficacy of clinical treatments. In the realm of recombinant protein production, these molecules can allow precise isolation and measurement of desired recombinant proteins from complex mixtures by selectively targeting specific protein tags or domains, ensuring the consistency and purity of protein products. Currently, antibodies are most commonly used affinity reagents in these fields but are limited by production complexity, batch variability, high cost, and low stability. Aptamers, known as ‘chemical antibodies’ but composed of nucleotides, are considered potential next-generation affinity reagents. Aptamers are obtained via a synthetic process, termed SELEX, of iterative affinity selection and polymerase chain reaction (PCR) amplification of target-binding members from a randomized oligonucleotide library. This process is traditionally labor and resource-intensive and time-consuming. In this thesis, microfluidic technology is employed to enable time-efficient and cost-effective generation of aptamers for monoclonal antibodies and recombinant proteins toward applications in therapeutic drug monitoring and quality control of recombinant protein production. This thesis starts with a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection (called “chip-selection SELEX”), and fully integrated microfluidic affinity selection and PCR amplification (termed “full-chip SELEX”). The comparison results indicate that chip-selection SELEX offers the lowest cost and highest efficiency in aptamer isolation. We then use chip-selection SELEX to streamline the process of isolating anti-idiotype aptamers targeting human monoclonal antibodies against spike protein of SARS-CoV-2 virus. The process is completed within only 5 rounds of SELEX within two days, which represented a significant improvement when compared to conventional methods whose completion generally requires more than 10 SELEX rounds in up to a month. These anti-idiotype aptamers are combined with a graphene-based affinity nanosensor to enable rapid antibody concentration measurements to inform therapeutic decisions in a timely manner. In addition, a microfluidic dual-aptamer sandwich assay with highly efficient isolation of aptamers is developed to enable rapid and cost-effective detection of tag-fused recombinant proteins. This approach addresses both the limitations of current dual-aptamer assays and commonly encountered difficulties in the lack of aptamers available for such assays, by first using chip-selection SELEX to generate aptamers and then employing these aptamers to implement a microfluidic dual-aptamer assay for quality control during recombinant protein production. Despite the high efficiency in aptamer isolation using chip-selection SELEX, the full-chip SELEX platform is still desired for minimal manual operation and reagent consumption. The current full-chip SELEX platform has low isolation efficiency and could not offer information of affinity selection process. Herein, by introducing asymmetric PCR into the full-chip SELEX process, we improve the efficiency in aptamer isolation and can successfully monitor the selection progress. This real-time monitoring capability allows us to identify the optimal point to terminate the SELEX process, preventing the potential loss of aptamer candidates and reducing the overall consumption of time and reagents. In addition, introducing solution phase-based asymmetric PCR addresses a notable technical challenge of on-chip PCR bead replenishment, toward complete automation of the full-chip SELEX platform. Furthermore, a holder equipped with connection pins is designed to enable the reversible connection between gold electrodes and electrical wires. This design promotes the reusability of gold electrode-deposited glass substrates, resulting in a substantial reduction in chip fabrication costs. In addition to the SELEX protocol development effort, we also present efficient and cost-effective microfluidic approaches for post-SELEX aptamer characterization, including aptamer identification and kinetic aptamer-target binding measurements. To mitigate the expensive and time-consuming nature of aptamer identification from SELEX-generated target-binding sequence pools, we present an approach that is based on a cost-effective and efficient procedure to generate modified single-stranded DNA copies of the aptamer candidates and then assess the affinity of the resulting modified ssDNA strands to target molecules. The approach is applied to identify aptamers from 12 candidates with consistent results, but at a cost three times lower than that of established methods. We also present a microfluidic fluorescence assay, which exploits a synergistic combination of microfluidic technology and fluorescence microscopy, to realize cost-effective and multiplexed measurement of kinetics of aptamer-target analyte binding without requiring special-purpose equipment.

The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.

This thesis presents an investigation of fluorescence resonance energy transfer (FRET) as a reporting signal for protein-protein interactions. Quantitative optical assays to measure protein binding, conjugation and deconjugation are developed and results validated by conventional biochemical techniques. The optical techniques developed provide fast, cheap, quantitative and accurate alternatives to conventional methods. Fluorescent protein fluorophores ECFP and Venus-EYFP were chosen as they are a non-interfering FRET pair and provide an inexpensive and convenient cloning-based labelling method. The small ubiquitin-like modifier SUMO and the SUMOylation pathway leading to its conjugation to target proteins is investigated as a model system. These assays are hence particularly relevant to research on post-translational modification and ubiquitin systems. In protein-protein binding assays we utilise both steady-state and time-resolved FRET detection to measure the equilibrium binding constant of the well-characterised pair SUMO1 and Ubc9. An assay in multi-well plate format is also presented, which uniquely enables repeat measurements under varying conditions and under the addition of further substances. The multi-protein binding interactions of the SUMOylation pathway including RanBP2 are analysed in binding inhibition assays. Our results clarify the role of RanBP2: a covalent SUMO1-Ubc9 link is required for the formation of a trimeric complex, although mutual binding sites are present on all three proteins. Furthermore, the binding of SUMO1 and Ubc9 is disrupted by RanBP2, which may be an essential step in transferring SUMO1 to its target protein. A FRET-based kinetic study of this conjugation process to RanGAP1 is presented. An assay to monitor the deconjugation of SUMO1 by specific proteases is established using a doubly-tagged SUMO construct. This enables a quantitative analysis of protease and substrate specificity based on real-time kinetic data, a characterisation of crude cell extracts and a high-throughput screen for protease inhibitors using FRET. A screen of the National Cancer Institute (NIC) diversity set for SenP1 inhibition reveals nine suitable compounds, which are potential anti-cancer drugs. The results of two further projects, the study of protein-protein binding by measuring small refractive index changes and the autofluorescence of normal and neoplastic cervical tissue models are also presented. In the latter, principal component analysis was used to systematically identify emission regions of significant variation between samples, enabling discrimination between healthy and pre-cancerous tissue models.