Protein purification is a critical step in the downstream processing of protein. Although chromatography is the most employed technique for protein purification, novel strategies that reduce operational costs and increase the amount of purified protein are being developed. These strategies have the possibility to reduce the price of protein-based pharmaceutical and biotechnological products through the reduction of purification cost. Preparative protein crystallization is one such economically-sustainable alternative to chromatography, however protein crystallization is very slow and is difficult to implement in current protein purification protocols. In our lab, we explore the use of metastable liquid-liquid phase separation (LLPS) to enhance protein crystallization. LLPS is typically induced by cooling protein aqueous samples below a well-defined temperature, called the LLPS temperature. We have previously shown that cooling aqueous samples of lysozyme in the presence of NaCl (0.15 M) and HEPES (0.10 M, pH 7.4) below LLPS temperature reproducibly produces yields of lysozyme crystallization above 90%. However, this process requires sample cooling to relatively low temperatures
(below –10 °C). In this poster, we examine the use of polyethylene glycol (PEG) as an additive that increases LLPS temperature. Our experimental results show that PEG increases LLPS temperature without appreciably altering formation of lysozyme crystals. The effect of PEG concentration on LLPS temperature is explained by considering the mechanism of macromolecular crowding.

The goal of this project is to develop synthetic riboswitches for trimethylamine N-oxide
(TMAO). TMAO has been shown to regulate various physiological processes involved in the
development of atherosclerosis. A riboswitch is a non-coding RNA molecule that specifically
binds to a ligand and thereby controls the expression of genes in the downstream. A synthetic
riboswitch for TMAO could be useful for regulating gene expression in response to TMAO and
detecting TMAO in complex biological samples such as urine and blood. The 17 nucleotides in
the aptamer domain of a naturally occurring glycine riboswitch were randomized to generate a
library containing billions of different variants. The library was placed in the upstream of the
cat-upp fusion gene for a series of dual genetic selections. The positive selection is done in the
presence of TMAO to identify functional riboswitches that make cells resistant to
chloramphenicol. The negative selection is performed in the presence of 5-fluorouracil to kill
the cells containing the riboswitch variants activated by any endogenous molecules. Once
identified by several rounds of dual genetic selections, the synthetic TMAO riboswitches will
be tested by colorimetric and fluorescence assays.

Chirality is a property of two molecules of the same composition to not be structurally superimposable on each other. Chiral structures are both common and essential throughout nature. Porous material can be used as templates for creating chirality. The use of cellulose as a template provides environmentally friendly alternatives for templating chirality onto materials such as silica. New research in this field includes using chiral silica templates for the synthesis of chiral perovskite films, a semiconductor material with bright light emission found in light sources like light emitting diodes (LEDs).

This project began by testing cellulose obtained from several different vendors to determine which product has the ideal properties for use in chiral films. An important aspect of cellulose product is its relative acidity/basicity (quantified in terms of pH), which can be regulated to control the degradation of cellulose into nanocrystals as well as the formation of chiral structures. Initially, the cellulose was acidified using sulfuric acid which causes aggregation and kinetic arrest within particles. A technique known as ion exchange chromatography is now being used in these experiments as the method of choice for acidifying cellulose samples.

The time allotted for cellulose nanocrystals to obtain chiral conformation is also an important aspect of creating chiral films. Initially, samples were not left to stand motionless and were immediately converted into silica films. Currently however, our procedure has been modified to allow the chiral cellulose nanocrystals at least seven days of sitting undisturbed before the addition of a silica precursor molecule, thereby facilitating the chiral product from the rest of the product mixture. Upon successful isolation of chiral cellulose-silica films, experiments will be initiated to template the formation of chiral light-emitting perovskite structures by infiltration.

Texas Christian University’s Chemistry Club has always made a conscious effort to interest and include first year students through events and volunteering. This year’s project was focused on boosting numbers and cultivating a stronger community within the Chemistry and Biochemistry Department by specifically holding events to engage first year students. Events included the citrus social, periodic table of cupcakes, murder mystery play, tie dye event, and jeopardy night. This gave students a chance to get to know their peers better, to interact with a wide range of professors, and to gain knowledge about chemistry from outside the classroom. The success of these events has lead to record high attendance not only for the events but also for the Chemistry Club meetings held bi-weekly. The success of this initiative will ensure these and other events will be hosted for years to come, hopefully growing to the size where other schools can join and collaborate, further building a community within the field of chemistry and biochemistry.

Predicting pKas is an outstanding challenge in computational chemistry. The Green group
at TCU is working to develop a library of pyclen derivatives that can successfully reduce oxidative
stress within the brain of people afflicted with neurodegenerative diseases1. Predicting the various
pKas of these flexible molecules, which are charged at neutral pH, challenges conventional
approaches to predicting pKas. For each pyclen derivative, we combine an extensive survey of
protonation site isomers, with conformational sampling using the CREST package2, DFT
calculations with continuum solvent models, followed by a linear fit to correct the solvent models
limitations for calculating energy of highly charged species. We can predict three to five measured
pKa values for each pyclen derivative with a RMSD of 0.9 pKa units, which is competitive with
the best-physics based method in the SAMPL6 blind challenge for the first pKa3. We are pushing
the boundaries of computational chemistry and its abilities to predict multiple pKas of flexible
molecules.