The Great Salt Lake in Utah is an important stopover point for many migratory bird species. Birds that stop to breed or forage at the Great Salt Lake may be at risk of mercury contamination due to high levels of methylmercury that are found in the lake. The purpose of this study was to examine the transfer of mercury from the lake into the terrestrial food web using organisms at the base of the food web. During the summers of 2019-2021 western spotted orb weaver spiders (Neoscona oaxacensis) and, when possible, brine flies (Ephydra sp.) were collected from various sites on Antelope Island. These specimens were analyzed for total mercury content using a Nippon MA-3000. In addition, satellite imagery and GIS software were used to document the approximate distance from the collection sites to the water surface. We examine differences between years, study sites and spider body size. We also examined the correlation between mercury levels and environmental conditions.

The Molding Melanin Magic mentorship program through TCU Pre-Health is geared to impact minority female student populations at the Texas Academy of Biomedical Sciences (TABS) in Fort Worth. The program provides small group mentorship as high school students are paired with a college student in their area of interest. Along with mentorship, workshops are utilized as a method of increasing confidence, exposure, and overall knowledge about college and STEM careers. By coupling workshops and mentorship, the Molding Melanin Magic program seeks to encourage mentees to serve as mentors along their educational journey, and apply for college and professional school to pursue a career in STEM.

Spiders are sentinel species, organisms that serve to map the bioavailable fraction of contaminants in an ecosystem by retaining their contaminants in their tissues. For example, spiders in the families Tetragnathidae and Araneidae are frequently used as sentinels of mercury contamination of aquatic ecosystems. Spiders are frequently preserved in alcohol prior to contaminant analysis but the impact of contamination on mercury concentrations in spiders has not been assessed. The objective of the present study was to determine the effects of different preservation methods on mercury concentrations in tissues of spiders in the families Tetragnathidae and Araneidae. The spiders were collected along water sources using nets and gloved hands. The Tetragnathids were collected from grassy terrain or a bridge overhanging the water of Lake Weatherford. The araneids were collected from a boat dock overhanging Eagle Mountain Lake. On site, each spider was placed into its respective bottle of varying ethanol or Ziplock's for freezing. Individual spiders were placed into one of three different concentrations of ethanol (100%, 95% , 70%) or frozen. Following about two months of preservation, the spiders were dried and run through the DMA-80 collecting the data for data analysis. Data xxxx *insert conclusion info*

Role of ClpX in the stress response and virulence of Bacillus anthracis: protease or chaperone?
Lillian Wilson, Vuong Do, S.M McGillivray
Department of Biology, Texas Christian University

Anthrax is a lethal infectious disease caused by the bacterial pathogen Bacillus anthracis. Our lab studies the virulence and antibiotic resistance of B. anthracis and we have identified a chromosomal gene clpX, as an important virulence factor, as its loss increases susceptibility to cell-envelope targeting antibiotics such as penicillin, daptomycin, and the antimicrobial peptide LL-37. ClpX is an ATPase that can act autonomously as a chaperone, or with a proteolytic core, ClpP, to degrade proteins. To investigate the mechanism ClpX uses, a plasmid pclpXI264E was designed with a mutation in clpX (I264E) that prevents ClpP binding and inhibits the formation of the ClpXP protease but does not disrupt the chaperone activity of ClpX. We used this to create 4 strains in the unencapsulated Sterne strain: wild-type and ∆clpX containing the empty inducible plasmid pUTE657, complementation plasmid with the non-mutated clpX gene (∆clpX + pclpX), and the mutated plasmid (∆clpX + pclpXI264E). Prior research done on these strains confirmed that ClpX relies on protease activity in antimicrobial stress; however, our goal was to assess its response in other environmental stressors such as acid stress, heat stress, and its virulence in vivo with the Galleria mellonella infection model. We find that that the protease activity of ClpX is important for all of these stresses. These results build on our earlier understanding and demonstrate that formation of the ClpXP protease is critical and any future development of drugs targeting the ClpX system should focus on protease formation rather than chaperone activity.

This research is focused on gaining a better understanding of PKC-epsilon a calcium-dependent protein kinase involved in a wide range of cellular functions including cell proliferation, survival, and apoptosis. The interest in PKC-epsilon derives from the discovery of a de novo mutation in the PKC-epsilon gene in patients suffering from SHORT syndrome. This syndrome is a debilitating disorder characterized by short stature, hyperextensibility, ocular depression, Rieger anomaly, and teething decay. The project involved recapitulating the naturally occurring de novo mutation in vitro as well as determining if other mutations in PKC-epsilon could cause similar disease-state phenotypes. Using a technique known as Site Directed Mutagenesis mutations were introduced into the PKC-epsilon gene and the effects of these mutations on the protein expression were assessed. This mutational analysis will help identify the regions of PKC-epsilon that are vital for its function. This will help elucidate the effect of the same mutations in patients and could help predict the severity of disease. Obtaining a clearer picture of the different regions of the PKC-epsilon protein allows for future studies to focus on successfully fixing these regions when they become damaged and could therefore be used to help patients with SHORT syndrome.

N-terminal acetylation is essential for the stability, activity, and targeting of proteins in eukaryotes. However, most eukaryotic proteins are not acetylated when expressed in bacteria. Therefore, it is of practical significance to control N-terminal acetylation of recombinant proteins in bacteria. RimJ is an N-terminal acetyltransferase (NAT) known to acetylate many recombinant proteins with a narrow substrate specificity in E. coli. This project is aimed to increase the applicability of RimJ for the N-terminal acetylation of a broad range of recombinant proteins.
Based on the AlphaFold-predicted structure of E. coli RimJ, we predicted that six amino acids (Y35, E46, R49, Y106, Y170, and L171) may recognize substrate proteins in the active site. We created RimJ variants, in which one or two of these amino acids are changed to alanine, a small neutral amino acid, so that the active site becomes larger to accommodate substrate proteins containing bigger N-terminal amino acid residues. The RimJ variants were created using site directed mutagenesis, confirmed by DNA sequencing, and co-expressed with Z domain mutants that were not acetylated by the wildtype RimJ. The Z domain mutants were isolated by immobilized metal ion affinity chromatography and analyzed by mass spectrometry for their N-terminal acetylation patterns.

In the past two centuries, tuberculosis (TB) has killed over 2 billion people. TB is an airborne contagious infection that usually attacks the lungs and can spread to the brain and spine. Today TB is treated with 6-12 months of antibiotics and if the medication is ended early the treatment is ineffective. There are also drug resistant forms of TB that are caused by mutations of the bacteria and this process is sped up by the overprescribing of antibiotics which is a growing problem. Dr. Jeffrey Aube created a drug that attacked both non replicating and replicating TB bacteria in the body. This was a major step from previous medicines that could only attack one. We are creating a library of TB drugs that are customizable, efficiently made, and easily purified. These customizable drugs will not only create a large range of effective medicines but also can treat TB that is resistant to antibiotics. Tuberculosis is still one the leading infectious disease killer today, claiming 1.5 million lives annually and we are making drugs that could change that and save millions of lives.

Pyridinophane molecules have recently been shown to have both antioxidant and pharmacological properties suitable for therapeutic applications targeting neurodegenerative diseases, including Alzheimer’s. We have synthesized derivatives of the parent molecules with substitutions on the pyridine ring (L1) or on the ‘side’ of the macrocycle (L2) designed to increase the antioxidant activity beyond that of the parent molecule in hopes of producing a molecule suitable for pharmacological testing in animal models. The lab is currently working towards substituting on the ‘bottom’ of the macrocycle (L3) to characterize and compare substitutions at each of the three positions.

Macrocycles are molecules containing at least one ring composed of 12 or more atoms. Macrocyclic drugs have been used clinically for decades. Many interfere with protein-protein interactions. Therapeutic intervention requires that macrocycles remain flexible to facilitate the adoption of different conformations. Specifically, small compact hydrophobic conformations are required to cross cell membranes. The ability of a macrocycle to perform these contortions is predicted by its octonal:water partition coefficient, its so-called logP. Macrocycles (as well as small molecule drugs) that are suitable for oral delivery have a logP value <5. In this study, methionine containing macrocycles are studied. The studies commence with the synthesis of a macrocycle with a dimethylamine auxiliary group that allows for solution-phase NMR analysis. Upon formation of the macrocycle, oxidation to sulfone and sulfoxide derivatives was executed. These macrocycles are of interest because the impact that oxidation has on log P values has not been reported. Additionally, S-oxidation could change the conformation of the molecules. Synthesis beings with substitution of trichlorotriazine with BOC-hydrazine, followed by treatment with methionine in basic conditions. The final substitution of the triazine installs the auxiliary group, dimethylamine (NMR). Amidation with 1,1-diethoxypropyl amine using a peptide coupling reagent yields the monomer. Cyclization using TFA yields the macrocycle. NMR spectroscopy confirms macrocyclization and gives insight into the solution conformation of the molecule. Oxidation strategies and the results of logP analysis will be developed.

Proper functioning of BRCA1 and PALB2 are essential in preventing tumor formation. Upon detection of DNA damage, BRCA1 binds to PALB2, leading to formation of the BRCA1-PALB2-BRCA2 DNA repair complex which is recruited to double-stranded break sites. Mutations in the genes coding for BRCA1 and PALB2 may disrupt this binding interaction, causing obstructions in DNA damage repair and increased breast cancer risk. Variants of unknown significance (VUS) found in breast cancer patients are genetic variants whose impact on the health of individuals are not yet known. Our study characterizes the effects of these VUS on the BRCA1-PALB2 binding interaction. Site-directed mutagenesis was used to generate BRCA1 and PALB2 VUS. It was found that the binding event between BRCA1 and PALB2 is enthalpic in nature and can be measured adequately via isothermal titration calorimetry (ITC). Thus, ITC was employed to identify whether the VUS disrupted binding. ITC data suggest that several PALB2 and BRCA1 VUS exhibit disruptions of the BRCA1-PALB2 binding interaction, but to varying degrees. We will share the data for variants tested thus far and emerging themes for prediction of the roles residues in both proteins play in the vital interaction.

Barriers to rotation within triazine compounds have been previously explored by Katritzky and Birkett [1-2], but these studies have been limited to differences in the substituent groups on the triazine as well as the degree of substitution (mono, di, tri). This study explores how the barriers to rotation within triazine containing compounds are affected by solvent and protonation state. Overall, these molecules are of interest due to their wide range of applications in dendrimer and macrocycle synthesis as well as pharmaceutical drug development [3-4]. The results of this study illustrate how solvent selection can significantly impact the distribution of rotational isomers (rotamers) and how barriers to rotation can be increased by protonation of the triazine ring.

Alzheimer’s Disease (AD) affects over 6.5 million Americans over the age of 65. Previous research links AD with the aggregation of Amyloid-beta-40 (AB40) in the brain, which creates neurotoxic plaques, causing further development of AD in the brain. A potential therapeutic mechanism in the treatment of AD is using drugs that will prevent the formation of these plaques, which is possible with Metal Chelation Therapy.
Metal ion chelation ideally stops metal ions from aiding in the aggregation of AB40. However, to deliver metal chelating agents to the brain, a drug-delivery mechanism is required that will be able to deliver this medicine across the Blood-Brain Barrier. Porous silica is a potential drug delivery material due to its particle size, high loading capacity, tunability, and biocompatibility. Along with these characteristics, porous silica can create a “sustained” release of a given drug, allowing for a slow and steady release profile, reducing the risks of medication side effects.
This project seeks to establish the optimal loading capacities of a class of potential AD therapeutic molecules known as pyclens into porous silica, each with different pyridyl moieties and chemical functionalities along the rim of the molecule. Encapsulation efficiencies measurements for these pyclen derivatives reveal loading percentages in the 10-19% range, varying by pyclen identity. Additionally, release studies monitored diffusion over time to find which pyclen molecule achieved “sustained” release. All loaded pyclen species were able to show sustained release after 20 minutes. Additional release studies of these molecules in the presence of copper (Cu2+) remain to be completed to ascertain the ability of release drugs in the presence of Cu2+ to inhibit AB40 aggregation, followed by independent assays of AB40 solubility under such conditions.

Two proteins, BRCA1 and PALB2 are known to aid in DNA damage repair through homologous recombination. Both proteins are phosphorylated upon DNA damage, and we hypothesize that the phosphorylation of these proteins acts as an “on switch” to allow the proteins to interact and form the DNA repair complex. To test this hypothesis, we mimicked phosphorylation on the BRCA1 protein to test the binding affinity between BRCA1 and PALB2. Phosphomimicking mutants are created by mutating an amino acid with the ability to be phosphorylated and acquire a negative charge, such as threonine (T) or serine (S), to a negatively charged amino acid, such as glutamic acid or aspartic acid. Recent research has shown that specific phosphorylation sites, such as T1394 in BRCA1 are essential to DNA damage and repair in cells. We have created a phosphomimic mutant in this specific T1394 site by mutating threonine to glutamic acid. We are currently measuring the effect that this mutation has on the ability of BRCA1 to bind to PALB2 in vitro. The obtained data will reveal whether phosphorylation has an impact on the interaction between these two proteins or not.

Metal-halide perovskites are crystalline materials that work as a semiconductor in both Light Emitting Diodes (LEDs) and solar cells. In general, perovskites possess the formula ABX3. For this project, the A site is an organic molecule such as Methylammonium (MA), the B site is Lead, and the X site is Bromide. While perovskites are easily fabricated, their crystal size and number of defects present are challenging to control. Defects cause LEDs to be less stable and/or less photoluminescent (bright) and cause solar cells to be less efficient at converting light to energy. One approach to reduce the number of defects is to use ionic liquids during perovskite formation. Ionic liquids are compounds made of ions in the liquid state due to a low melting temperature. They can be added to the perovskite precursor solution to slow down the crystallization process so that fewer defects are created. The goal of this project is to create new metal halide perovskites in the presence of selected ionic liquids, evaluate their structure and photophysical properties, with the long-term goal of creating new LEDs that are both stable and efficient.

In this project, cetyl-ionic liquids (cetyl meaning 16 carbon chains) were investigated for their effects on perovskite structure and light emission. The three ionic liquids were investigated: [C16-mim]Br (referred to as "IL1"), [C16-py]Br ("IL2"), and [C16-C1pyrr]Br ("IL3"). Variations on the addition method of ionic liquids to the perovskite precursor were studied as well. It was hypothesized that the inclusion of cetyl-ionic liquids will protect the perovskite films from the environment (increasing stability) by providing a hydrophobic layer on the surface and will improve the electronic properties by filling in pinholes that cause defects. It is found that perovskite films with IL3 are more photoluminescent than the perovskite films formed with IL1, IL2, or no IL (control). Preliminary experiments varying the addition method of IL3 during film formation have shown that the perovskite films are brightest when IL3 is added to both the precursor and the antisolvent layers at the beginning of the fabrication process. These results, along with detailed structural characterization of a given perovskite film, will be discussed in this presentation.

To fight disease, pharmaceutical companies have historically prepared small molecules designed to interfere with specific sites on proteins (enzymes) to prevent chemical reactions from taking place. However, a second paradigm for interfering with proteins has gone largely unexplored--blocking protein-protein interactions. To accomplish the latter, large molecules are needed to bind to large areas on the protein target. However, large molecules present additional challenges. Typically, they are hard to synthesize, not orally available, and typically cannot cross cell membranes. Nature has designed large molecules like cyclosporin that should not work as drugs based on our current understanding. Despite its size, cyclosporin is orally available and can cross cell membranes. This research explores the design, synthesis, and conformational analysis of similar large ring-shaped molecules, so-called macrocycles. In this work, we are increasing the size of the ring-shaped molecule. By increasing the size of the ring-shaped molecule and varying the amino acid (in this case, valine), we are expanding the possible ways in which our macrocycle may interfere with protein-protein interactions. Here, a 26-atom macrocycle is reported. 1H NMR spectroscopy reveals a protonated molecule that is highly dynamic which has access to a beta-sheet conformation.

Macrocyclic drugs adopt multiple conformations--a behavior referred to as chameleonicity--to navigate hydrophobic cellular membranes and aqueous intracellular environments. The rules for understanding this behavior are beginning to emerge through studying existing drugs and the synthesis of model systems. Historically, one challenge to macrocycle synthesis is low yield reactions. To this end, dynamic covalent chemistry has been explored. Here, macrocycles are afforded readily by dimerization with the formation of two hydrazones.

The efficiency of the macrocyclization reaction led to the hypothesis that upon formation of the first hydrazone, the acyclic intermediate was preorganized to place the hydrazine and acetal in close proximity thereby reducing the likelihood of oligomeric or polymeric products. The preorganization could result from a network of hydrogen bonds. Moreover, in an acidic environment, wherein the triazine ring is protonated, the opportunity for bifurcated hydrogen bonds emerge. Computation has been used to identify sites for protonation and the energetic contributions of hydrogen bonding.

To explore templating and the role of protonation in the formation of hydrogen bonds, model systems were prepared that emulate ‘half’ of the macrocycle. The acetylated aminoacetal offers a well-resolved NMR spectrum. In contrast, hindered rotation about the triazine-N bond leads to a mixture of rotamers in the hydrazine component. However, upon condensation, a single rotamer is observed and resonances corresponding to the hydrogen bonded protons emerge downfield between 7-12 ppm. Computation provides estimates of the energetic contribution of the bifurcated hydrogen bond as well as the hydrogen bond formed in the absence of protonation. The results of titration and variable temperature NMR experiments will also be described.

The mis-regulation of reactive oxygen species and transition metal ions contributes to the onset of Alzheimer’s Disease. Reactive oxygen species are a natural byproduct of metal redox cycling that occurs within the body and are important in processes like homeostasis and various pathways of cell signaling. Two series of pyridinophane ligands were produced and evaluated for the ability to target the molecular features of Alzheimer’s Disease. The functionalized pyridinophanes were chosen to analyze their blood-brain barrier permeability and radical scavenging ability when included within a molecular scaffold. Preliminary results with the DPPH assay indicated a significant increase in radical scavenging activity for ligands containing electron-donating substitutions in comparison to the parent ligands. These results warrant further exploration into the mechanism of the activity observed.

Petroleum crude oil, unconventional crudes, and renewable bio-crudes are essential materials in our everyday lives. They fuel vehicles, heat buildings, provide electricity, and are used to produce a multitude of other materials, such as plastics and solvents. Crudes are highly complex chemical mixtures, estimated to contain between 100,000 and 100,000,000,000,000,000 unique molecules. Since 2015, single-molecule imaging has visualized hundreds of chemical structures, and historical literature has published thousands of proposed structures. This project builds an open database populated with published crude structures enabling data-driven analysis of these structures, and detailed workflows, allowing for easy future insertion of new molecules into the database. This database can be used to make calculations and predict characteristics of molecules, such as viscosity, density, and reactivity, which are all critical in refinery plants, transportation, and usage of these fuels. Performing queries on the molecules in the database to filter for specific characteristics allows scientists to develop more successful experiments by refining their hypotheses to account for the query results displaying possibilities of their desired outcome.  

This research aims to understand how to design and control molecular hinges. The molecular hinges of interest are nano-sized equivalents of door hinges. Such hinges could find applications in new materials or the design of new drugs.

The foundation for this research was the observation that a large, ring-shaped molecule - a so-called macrocycle – prepared by a colleague folded and unfolded rapidly at room temperature. Two research questions arose from this observation: was the hinge behavior unique to this molecule, and could the hinging rate be controlled?

Addressing these questions required the three-step synthesis of a related macrocycle. This new molecule had groups equivalent to putting grit around the hinge's pin. The difference in the rate of hinging motion due to the addition of these groups was observed using a technique called variable temperature NMR spectroscopy.

The results of this work revealed that hinging is a general phenomenon for some of these macrocycles. Second, the 'molecular dirt' designed into this new hinge reduced the rate of hinge motion from 2000 times per second to 20 times per second.

This work is being written up for communication to the Journal of the American Chemical Society based on the novelty of this molecular device and the scientific community's interest in molecular machines.

Dating back to 1550 B.C., ancient civilizations used moldy bread and medicinal soil to treat infections and wounds. Today, antibiotics are commonly used to treat bacterial infections. Salvarsan, the first antibiotic, was developed in 1910, followed by penicillin in the late 1920s. However, the widespread use of antibiotics and limited research has resulted in the emergence of antimicrobial resistance, posing a global threat. To address this, developing new antibiotics is crucial. Vancomycin, a potent antibiotic isolated in 1955 and synthesized in the late 1990s, is a target for this purpose. Despite its effectiveness, vancomycin is challenging to produce, with yields not exceeding 5%. Thus, this project aims to create a structure in four steps, with a yield greater than 50% that resembles vancomycin’s iconic 3-D bowl shape.

Platinum compounds play an important role as anticancer agents. Their ability to bind to DNA in the nucleus (by a process known as intercalation within DNA base pairs) result in DNA damage and cell death. Unfortunately, these platinum-containing compounds lack specificity toward cancer cells and attack normal healthy cells that results in significant side effects as a consequence (loss of hair, nausea, among others).
Our group has developed a method to incorporate platinum on the surface of our silicon Nanotubes using (3-Aminopropyl) triethoxysilane (APTES) as a functional arm to the Nanotubes. The Silicon nanotubes have attracted great attention in applications relevant to diagnosis and therapy, owing in part to its biocompatibility and biodegradability in cells.
Once inside the cell, platinum is released slowly, thus allowing an interaction with DNA. Our previous results using this technology showed significant toxicity on a type of cancer cell known as HeLa. While these findings are promising, specificity has not yet been achieved.
Cancer activates signaling pathways that translates on overexpression of specific proteins/receptors. Particularly, folate receptors (FR) are present in 90-98% of ovarian, prostate, uterus, breast, as well as some adenocarcinomas. FR expression is very limited in normal cells and generally not accessible to blood flow which makes it a suitable and promising system to target cancer. These receptors are glycopolypeptides that present high affinity for folic acid (FA).
A viable strategy has been identified, involving the conjugation of a molecule known as glutathione to act as a linker to the surface of the silicon-based platinum nanoparticles through N-Hydroxysuccinimide (NHS) activation, followed by substitution with folic acid.
The cellular evaluation of this material shown high cytotoxicity against Hela cells and selectivity, in compare with material without Folate.

Peptidomimetic macrocycles are of ever-growing interest to the field of pharmacology as candidates for inhibiting supposed "undruggable" sites (such as protein-protein interactions). An important property of pharmacophores within drug development is the partition coefficient (often expressed as logP or logD), which measures the ability of a molecule to partition between aqueous and organic media, effectively expressing the ability for a drug to diffuse into a cell from the bloodstream. Our group has previously synthesized several amino acid-containing triazine macrocycles through facile three-step procedure yielding folded, sometimes dynamic, macrocycles in good yields. With twelve macrocycles, a trend in logD values has emerged, allowing for the rapid prediction of the macrocyclic conformation per its respective logD values. Each macrocycle is folded, but the extent of triazine-triazine overlap, side chain van der Waals interactions, and shielding of its central proton is reflected in the divergence of the macrocycle's logD from a central trendline. The ability to predict the macrocycle's logD values via additive, atomistic, algorithms is also shown to reveal this divergent trend. Structures of these triazine macrocycles were elucidated through proton and nOesy/rOesy NMR.

In the world of drugs, the chemical property that is most important is logP, the predictor of whether a drug can be taken orally and cross the cell membrane. Pharmaceutical companies will not explore molecules with logPs that are outside the ideal range. But what if predictions are wrong? The rules for predicting logP are based on small molecules, but the industry is moving towards large molecule drugs. This poster looks at synthesizing models of large molecule drugs (ring-shaped molecules called macrocycles) to determine if the logP of large molecules can be predicted. Synthesis of a hydrophobic macrocycle shows that the industry predicted logP failed. New prediction methods are needed. To develop these methods, additional macrocycles were made to serve as models for prediction. These molecules also allow us to explore another avenue in drug design challenge another paradigm in drug discovery. Pharmaceutical companies avoid hydrophilic functional groups because of ill predictions about logP. Combining these hydrophilic groups with predictable hydrophobic groups will make the molecule's logP acceptable. That is, by design, the undesirable hydrophilic group is balanced with the desirable hydrophobic group to bring polar groups through the membrane. Overall, the work will allow for a wider range of molecules to be considered for potential drug design.

Oxidative stress occurs when there is an imbalance between free radical activities, including those of reactive oxygen species (ROS), and the body’s natural antioxidant mechanism. To help restore this balance, the Green research group at TCU has developed tetradentate pyridine-containing cyclen macrocycles capable of simultaneously carrying out various modes of antioxidant activities. As drug candidates , these molecules need to be further modified with different functional groups to fine-tune their activities and pharmacological properties, resulting in a large library of up to hundreds of derivative structures. Isoelectric point (pI) and acidity (pKa) play a vital role in assessing the membrane permeability of these molecules. Given the size of the library, experimental determination of these values is an unnecessarily time-consuming endeavor. Using the state-of-the-art Density Functional Theory (DFT), this project aims to 1) show how pI values of any molecules in this library can be predicted with reference to a desired value and 2) predict the pKa of different acidic sites on these multifunctional molecules. This can potentially shed light on the effects of covalent modifications on pI and pKa values, and with further optimizations, can be applied to a virtual screening protocol for any libraries of drug candidates.

Protein crystallization is regarded as a more economically sustainable strategy for achieving protein purification compared to traditional downstream processing chromatography. However, protein crystallization is not a well understood process and still relies on empirical protocols. This work examines the rational design of protein crystallization for lysozyme, a model protein, by exploiting the formation of metastable protein-rich droplets by liquid-liquid phase separation (LLPS). Specifically, sodium chloride, which is a salting-out agent, is used to induce LLPS, while 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) is a salting-in agent used to modulate LLPS conditions. It was found that HEPES enhances protein crystallization from protein-rich droplets. This effect can be explained by examining the relative shift of the LLPS boundary with respect to crystal solubility in the temperature-composition phase diagram. This work suggests that LLPS-mediated protein crystallization may be enhanced in the presence of salting-in agents.