Biochemistry, Biophysics, and Structural Biology Commons^™

Computational Identification Of Protein Catalytic Sites: Tests, Validation, Dan Kirshner, Jerome Nilmeier, Felice Lightstone

STAR Program Research Presentations

This project is one element of the analysis “pipeline” to characterize an organism that previously has not been well-studied. Once a protein of unknown structure has been computationally modeled (based on its sequence similarity to proteins with solved structures), then catalytic sites are identified on the model by comparison to a library of known sites. This work tested the identification algorithms with a set of proteins that have known structures and catalytic sites.

Thermal-Initiated Hydroxyethyl Methacrylate Functionalization Of Multiwalled Carbon Nanotubes, Greg Curtzwiler, Philip Costanzo, Ray Fernando, Jeffrey E. Danes, Keith Vorst

Chemistry and Biochemistry

Multiwalled-carbon nanotubes (MWCNTs) were functionalized via thermoinitiated free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) using benzoyl peroxide. Tip sonication was used during the polymerization reaction to separate agglomerated nanotubes. The functionalization was confirmed by control experiments and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Differential scanning calorimetry indicated that the addition of poly(HEMA)-MWCNTs to a two-component polyurethane coating will have little effect on the glass transition temperature of the coating. The poly(HEMA)-functionalized MWCNTs formed large colloidal structures of highly dispersed nanotubes in both the nonsheared and sheared coatings as determined by atomic force microscopy. This study determined a quick …

Direct Imaging Of Two-State Dynamics On The Amorphous Silicon Surface, S. Ashtekar, Gregory E. Scott, J. Lyding, M. Gruebele

Chemistry and Biochemistry

Amorphous silicon is an important material, amidst a debate whether or not it is a glass. We produce amorphous Si surfaces by ion bombardment and vapor growth, and image discrete Si clusters which hop by two-state dynamics at 295 K. Independent of surface preparation, these clusters have an average diameter of ~5 atoms. Given prior results for metallic glasses, we suggest that this cluster size is a universal feature. The hopping activation free energy of 0.93 ± 0.15 eV is rather small, in agreement with a previously untested surface glass model. Hydrogenation quenches the two-state dynamics, apparently by increasing surface …

Determining A Method For Rendering Low Cost Cdse(Zns) Core(Shell) Quantum Dots Aqueous Soluble Via Amphiphilic Polymer Wrapping, Patrick Mcbride

Materials Engineering

Herein is described the procedure of two amphiphilic polymer wrapping techniques that may be employed for obtaining aqueous soluble quantum dots (QDs) for use in biological fluorescent imaging applications. The advent of QDs has led to new nanoscale fluorescent materials that exhibit unparalleled quantum yields (QYs), high resistance to photobleaching, tunable emissions, and
absorption over a large optical range. However, the QD synthesis employed here at Cal Poly to obtain bright, photostable CdSe(ZnS) core(shell) QDs involves the use of organic solvents and surfactants, leading to hydrophobic QDs. Since all of biology relies on aqueous solubility, this hydrophobicity creates a major …

Oxidation Kinetics Of Silver Thin Films For Antimicrobial Applications In The Health Care Industry, Sean D. Morham

Materials Engineering

To determine if varying city water qualities from around the United States can support a developing “oxidation corrosion of silver thin films” method of creating aqueous ionic silver solutions for the anti-microbial treatment of healthcare linens, a test was devised holding all variables constant with the exception of water quality parameters. This corrosion style test was done in order to assess a pass/fail criteria of 25 ppb ionic silver concentration in the aqueous solutions which resulted from the corrosion of silver thin film coated substrates. Above 25 ppb being a pass, below 25 ppb being a fail. While varying the …

Performance Screening Of Chemostat Adapted Recombinant Zymomonas Mobilis Strains, Deirdre M. Beard, Nancy Dowe

STAR Program Research Presentations

Corn stover biomass can be pretreaed and hydrolyzed into soluble sugars to be fermented by microorganisms to ethanol. NREL has developed a recombinant bacteria Zymomonas mobilis 8b that metabolizes both five and six carbon sugars. During pretreatment, toxic inhibitors such as furfural and acetate are produced. NREL has made an attempt to adapt two sub-strains of Z. mobilis 8b to acetate and furfural by using a chemostat method. During the chemostat process, cultures were frozen back in glycerol and saved. In this study, those frozen cultures were revived and analyzed for performance in environments with varying concentrations of furfural and …

Caffeine Metabolism And Cytochrome P450 Enzyme Mrna Expression Levels Of Genetically Diverse Inbred Mouse Strains, Neal Addicott, Michael Malfatti, Gabriela G. Loots

STAR Program Research Presentations

Cytochrome P450 (CYP) enzymes activate or eliminate medications and other xenobiotics, and Caffeine has been used as an indicator of activity for some CYPs. The natural genetic variation in the human population for caffeine metabolizing CYPs does not yet have an established animal model for comparison, but the natural variation in inbred mouse strains presents an attractive starting point. The project gave four different strains of mice a 40mg/kg dose of caffeine and sampled blood and liver tissue after 30 minutes. Samples were analyzed using High Performance Liquid Chromatography (HPLC), reverse transcription PCR (rtPCR), and quantitative PCR (qPCR). The DBA/2J …

Microfluidic Paper-Based Analytical Devices: From Pocket To Paper-Based Elisa, Andres W. Martinez

Chemistry and Biochemistry

Microfluidic paper-based analytical devices (microPADs) began as a simple idea with an ambitious goal. The idea was to make microfluidic devices out of paper instead of plastic or glass. The goal was to develop low-cost and portable paper-based diagnostic devices to improve healthcare in developing countries. Over the past 6 years, many developments have been made in the emerging field of paper-based microfluidic devices. Reviewing the development of these devices in the Whitesides group at Harvard University (Cambridge, MA, USA) can provide some insight into the future of the field and encourage scientists from a variety of backgrounds to contribute …

Better Biomolecule Thermodynamics From Kinetics, Kiran Girdhar, Gregory Scott, Yann R. Chemla, Martin Gruebele

Chemistry and Biochemistry

Protein stability is measured by denaturation: When solvent conditions are changed (e.g., temperature, denaturant concentration, or pH) the protein population switches between thermodynamic states. The resulting denaturation curves have baselines. If the baselines are steep, nonlinear, or incomplete, it becomes difficult to characterize protein denaturation. Baselines arise because the chromophore probing denaturation is sensitive to solvent conditions, or because the thermodynamic states evolve structurally when solvent conditions are changed, or because the barriers are very low (downhill folding). Kinetics can largely eliminate such baselines: Relaxation of chromophores, or within thermodynamic states, is much faster than the transition over activation …

The Proteomic Response Of Sea Squirts (Genus Ciona Congeners) To Heat Stress: Evidence For Differential Thermal Sensitivities, Loredana Serafini

Master's Theses

The sea squirts Ciona intestinalis and C. savignyi have disparate distribution patterns, which may result from differences in their thermal tolerance limits. Because C. intestinalis, an almost cosmopolitan species, has a more widespread distribution, it is thought that it is better adapted to endure a wide range of temperatures. In order to compare the heat stress response between these two congeners, we studied global changes in protein expression, using a proteomics approach. To characterize the response to extreme heat stress, animals of both species were exposed to temperatures of 22°C, 25°C, and 28°C for 6 h, and then were …

Dual Inhibition Of Sodium-Mediated Proton And Calcium Efflux Triggers Non-Apoptotic Cell Death In Malignant Gliomas, William Harley, Candace Floyd, Tamara Dunn, Xiao-Dong Zhang, Tsung-Yu Chen, Manu Hegde, Hasan Palandoken, Michael H. Nantz, Leonardo Leon, K.L. Carraway Iii, Bruce Lyeth, Fredric A. Gorin

Chemistry and Biochemistry

Malignant glioma cells maintain an elevated intracellular pH (pH_i) within hypoxic–ischemic tumormicroenvironments through persistent activation of sodium–proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH_i of malignant gliomas by inhibiting isoform 1 of sodium–proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium–calciumexchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and …

Genotype And Breed Trend Influences On Citric Acid And Coagulation Times Of Raw Milk, Melissa Looney

Dairy Science

The objective of the study was to determine if citric acid levels measured in milk was related to genetic variants identified in Holstein and Jersey cows. The data used were milk samples collected from both Holstein and Jersey cows at Cal Poly, San Luis Obispo. Citric acid levels and other constituents were measured using FTIR methods with the FOSS MilkoscanTM FT2 on each sample. Genotypes were obtained for the DGAT 1 locus using polymerase chain reaction and an enzymatic digestion using the MWO I restriction enzyme. Observations were obtained on 13 Holsteins and 12 Jersey cows. Results indicated that citric …

Solving The Low Dimensional Smoluchowski Equation With A Singular Value Basis Set, Gregory E. Scott, Martin Gruebele

Chemistry and Biochemistry

Reaction kinetics on free energy surfaces with small activation barriers can be computed directly with the Smoluchowski equation. The procedure is computationally expensive even in a few dimensions. We present a propagation method that considerably reduces computational time for a particular class of problems: when the free energy surface suddenly switches by a small amount, and the probability distribution relaxes to a new equilibrium value. This case describes relaxation experiments. To achieve efficient solution, we expand the density matrix in a basis set obtained by singular value decomposition of equilibrium density matrices. Grid size during propagation is reduced from (100–1000) …

Novel Polymer Coupling Chemistry Based Upon Latent Cysteine-Like Residues And Thiazolidine Chemistry, Jospeh S. Carlson, Megan R. Hill, Taiga Young, Philip Costanzo

Chemistry and Biochemistry

Chain end functional polymers were prepared via reversible addition–fragmentation transfer (RAFT) polymerization techniques that were further chain extended with acrylonitrile. Under reducing conditions, latent cysteine-like residues were exposed at the chain ends. A variety of reduction conditions were explored and base polymers were then tethered together via thiazolidine chemistry.

Growth Hormone Alters Components Related To Differentiation, Metabolism And Milk Synthesis And Secretion In Mac-T Cells, Tasha Lynn Johnson

Master's Theses

The mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin and is extensively used to study bovine mammary epithelial cell function. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and induce cytoskeletal rearrangement in 3T3 fibroblast cell line and a Chinese hamster ovary (CHO) cell line. Few studies have focused on identifying the mechanisms involved in differentiated MAC-T cells’ response to GH. We tested the hypothesis that MAC-T cells would respond directly to GH and that …

Poisonous Rangeland Plants In San Luis Obispo County, Sara Litten, Amanda Ou

Animal Science

Poisonous Rangeland Plants in San Luis Obispo County is a comprehensive educational guide to rangeland plants that are toxic to domestic livestock. This guide begins with an exploration of how the biological systems are affected by the poisonous plant toxins. The biochemistry behind these toxins is included in the discussion. Next, reference material for fourteen plants that inhabit San Luis Obispo County is provided. This information includes specific toxins found in poisonous plants, affected animals, symptoms of poisoning, stages of growth, lethal dose, and distribution of the plant in California. This section of the guide is filled with helpful photos …

Overexpression And Partial Purification Of Recombinant Human Serine Hydroxymethyltransferase: A Potential Anticancer Target, Suprit Singh Deol

Chemistry and Biochemistry

Serine Hydroxymethyltransferase (SHMT) is a member of the thymidylate synthase cycle that provides pyrimidine nucleotides to actively dividing cells, including cancer cells. The other two enzymes involved in this cycle have been targets for clinical anticancer drugs, suggesting that inhibitors of SHMT may also be used as clinical anticancer drugs. In order to test potential inhibitors against SHMT, human SHMT has been overexpressed in E. coli and partially purified by selective ammonium sulfate fractionation followed by anion-exchange fast protein liquid chromatography. A coupled assay using L-allo-threonine and alcohol dehydrogenase was used to identify active fractions, which were analyzed …

A Natural Missing Link Between Activated And Downhill Protein Folding Scenarios, Feng Liu, Caroline Maynard, Gregory E. Scott, Artem Melnykov, Kathleen B. Hall, Martin Gruebele

Chemistry and Biochemistry

We propose protein PTB1:4W as a good candidate for engineering into a downhill folder. PTB1:4W has a probe-dependent thermal unfolding curve and sub-millisecond T-jump relaxation kinetics on more than one time scale. Its refolding rate in denaturant is a non-linear function of denaturant concentration (curved chevron plot). Yet at high denaturant concentration its unfolding is probe-independent, and the folding kinetics can be fitted to a single exponential decay. The domain appears to fold via a mechanism between downhill folding and activated folding over several small barriers, and when denaturant is added, one of these barriers greatly increases and simplifies the …

Electrochemical Sensing In Paper-Based Microfluidic Devices, Zhihong Nie, Christian A. Nijhuis, Jinlong Gong, Xin Chen, Alexander Kumachev, Andres W. Martinez, Max Narovlyansky, George M. Whitesides

Chemistry and Biochemistry

This paper describes the fabrication and the performance of microfluidic paper-based electrochemical sensing devices (we call the microfluidic paper-based electrochemical devices, μPEDs). The μPEDs comprise paper-based microfluidic channels patterned by photolithography or wax printing, and electrodes screen-printed from conducting inks (e.g., carbon or Ag/AgCl). We demonstrated that the μPEDs are capable of quantifying the concentrations of various analytes (e.g., heavy-metal ions and glucose) in aqueous solutions. This low-cost analytical device should be useful for applications in public health, environmental monitoring, and the developing world.

Programmable Diagnostic Devices Made From Paper And Tape, Andres W. Martinez, Scott T. Phillips, Zhihong Nie, Chao-Min Cheng, Emanuel Carrilho, Benjamin J. Wiley, George M. Whitesides

Chemistry and Biochemistry

This paper describes three-dimensional microfluidic paper-based analytical devices (3-D μPADs) that can be programmed (postfabrication) by the user to generate multiple patterns of flow through them. These devices are programmed by pressing single-use ‘on’ buttons, using a stylus or a ballpoint pen. Pressing a button closes a small space (gap) between two vertically aligned microfluidic channels, and allows fluids to wick from one channel to the other. These devices are simple to fabricate, and are made entirely out of paper and double-sided adhesive tape. Programmable devices expand the capabilities of μPADs and provide a simple method for controlling the movement …

Millimeter-Scale Contact Printing Of Aqueous Solutions Using A Stamp Made Out Of Paper And Tape, Chao-Min Cheng, Aaron D. Mazzeo, Jinlong Gong, Andres W. Martinez, Scott T. Phillips, Nina Jain, George M. Whitesides

Chemistry and Biochemistry

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based ―stamp‖) as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimetre. This method provides a new tool to pattern biochemicals—reagents, antigens, proteins, and DNA—on planar substrates. The …

Overexpression And Partial Purification Of Serine Hydroxymethyltransferase, Kimberly Rose Stevens

Chemistry and Biochemistry

No abstract provided.

Temperature Influence And Heat Management Requirements Of Microalgae Cultivation In Photobioreactors, Thomas Hagen Mehlitz

Master's Theses

Microalgae are considered one of the most promising feedstocks for biofuel production for the future. The most efficient way to produce vast amounts of algal biomass is the use of closed tubular photobioreactors (PBR). The heat requirement for a given system is a major concern since the best algae growth rates are obtained between 25-30 °C, depending on the specific strain. A procedure to determine temperature influence on algal growth rates was developed for a lab-scale PBR system using the species Chlorella. A maximum growth rate of 1.44 doublings per day at 29 °C (optimal temperature) was determined. In addition, …

Three-Dimensional Microfluidic Devices Fabricated In Layered Paper And Tape, Andres W. Martinez, Scott T. Phillips, George M. Whitesides

Chemistry and Biochemistry

This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D …

Alkoxyamine Polymers: Versatile Materials For Surface Ligation Applications, Lindsey Hines, Jessica Moore, David Dahl, Belinda Lady, Johnathan Brantley, Hasan Palandoken

Chemistry and Biochemistry

Immobilization of biomolecules (i.e., proteins, carbohydrates), on polymeric surfaces has been an area of intense research. The resultant bioconjugates often display increased stability, bioavailability and activity. Our research program seeks to explore the utility of the alkoxyamine (RONH2) functional group in new materials as versatile ligating sites for the immobilization of various compounds.

The ease with which alkoxyamines (RONH2) condense with aldehydes or ketones has prompted their widespread use in labelling liposome, bacterial and mammalian cell surfaces as well as chemoselectively ligating small molecule ‘recognition elements' onto polyfunctional substrates. These condensation reactions proceed in aqueous media to afford the robust …

Cariporide Prodrugs: Targeting Brain Cancer Cells Through Sodium-Proton Exchange Inhibition, Jacob Vervynckt, Johnathan Brantley, Jessica Moore, Mark Graves Ii, William Harley, Fredric A. Gorin, Hasan Palandoken

Chemistry and Biochemistry

More than 200,000 people in the United States are diagnosed with a primary or metastatic brain tumor annually. The life expectancy for these individuals is approximately 9-12 months from the time of diagnosis. This poor prognosis is due to the ineffectiveness of existing therapies (i.e., chemotherapy and radiotherapy) against brain cancer, where the primary problem is the inability to differentiate cancer cells from healthy brain cells.

Relative to healthy brain tissue, the heightened metabolism of cancer cells increases their reliance on the ion transport proteins NHE (sodium-proton exchanger) and NCX (sodium-calcium exchanger). Inhibition of these proteins disrupts the intricate pH …

Removal Of Benzaldehyde From Methanol Using Alkoxyamine Functionalized Silica Gel, Dino Sulejmanovic, Carrie Jo Pruitt, Eric Conte, Hasan Palandoken, Shing-Yi Suen

Chemistry and Biochemistry

Alkoxyamines (RONH₂) react readily with aldehydes and ketones to form stable oxime ethers, and, thus are expected to be excellent scavengers of these compounds. We installed alkoxyamines on silica gel surface to remove benzaldehyde from methanol. Silica gel was immersed in H₂O₂/H₂SO₄ solution to activate the surface. Activated silica gel was then reacted with 3-chloropropyltrichlorosilane. Displacement of the chloride with N-hydroxyphthalimide and subsequent treatment with hydrazine provided alkoxyamines on the surface of silica gel. The resulting silica gel surface with alkoxyamine functional groups was immersed in a 100 ppm benzaldehyde solution …

The Effects Of A Networked Data Acquisition System On Student Learning In General Chemistry Laboratories, Stuart C. Burris, Les L. Pesterfield, Darwin Dahl, Hasan Palandoken, Jacqueline Pope-Tarrence

Chemistry and Biochemistry

We have recently incorporated a networked data acquisition and analysis system into in our second semester general chemistry laboratories. Our investigation team has conducted a study to evaluate the effects of the networked data acquisition system on student performance on a Beer's Law laboratory. We focused on two specific phenomena: the effect of the networked data acquisition system on 1) student learning of specific content material and 2) student comfort level with laboratory work and data analysis. The study involved a networked data acquisition group (employing a UV-Vis spectrometer and colorimeters) and a control group (employing traditional, single wavelength spectrometers). …

Progress Toward Cariporide Analogs For Sodium-Proton Exchange Inhibition, Jessica Moore, Mark Graves Ii, Jacob A. Vervynckt, William Harley, Fredric Gorin, Hasan Palandoken

Chemistry and Biochemistry

The sodium proton exchanger (NHE) is particularly important in maintaining the intracellular pH in human heart and brain. Under anaerobic conditions (i.e., ischemia), a shift from oxidative to nonoxidative glycolysis occurs. The resultant decrease in the intracellular pH activates NHE, which increases the intracellular sodium, initiating the sequence of physiological events that lead to cell death. Thus, there has been great interest in the development of compounds that inhibit NHE. Indeed, potent NHE inhibitors are available. However, a fundamental impediment to the field is the delivery of these compounds to poorly vascularized tissues during the early phases of ischemia when …

The Effects Of Pc-Based Laboratories On Student Learning, Lester L. Pesterfield, Stuart Burris, Darwin B. Dahl, Hasan Palandoken, Jacqueline Pope-Tarrence

Chemistry and Biochemistry

The Department of Chemistry at Western Kentucky University recently incorporated a net-worked MeasureNet data acquisition and analysis system into in the second semester General Chemistry laboratories. Our investigation team has conducted a study to evaluate the effects of the MeasureNet system on student learning and performance on a Beer's Law laboratory. We focused on two phenomena: the effect of the MeasureNet system on 1) student learning of specific content material and 2) student interest in chemistry and science in general. The study involved a MeasureNet system group (employing a UV-Vis spectrometer and colorimeters) and a control group (employing traditional Spectronic …