Stability Studies on His420Phe Mutation in the Beta-Helix Domain of P22 Tailspike Gregory G. Abendroth, Matthew J. Gage, and  Anne Skaja Robinson Department of Chemical Engineering Correct folding is critical for protein function, and aggregates resulting from misfolding cause numerous human diseases. The insoluble nature of protein aggregates necessitates the use of model systems, such as the P22 tailspike, for examining the factors that affect aggregation.  P22 tailspike is a protein comprised primarily of beta strands in a beta-helix motif. The role of histidine 420 in P22 tailspike was examined by mutating H420 to phenylalanine (H420F) using Stratagene’s QuikChange Site-Directed Mutagenesis Kit. This mutant was chosen for study to examine the effect of charge on folding of the beta helix domain. The H420F mutant formed a trimer during expression in E. coli and was purified by anion exchange and hydrophobic affinity column chromatography. Chemical denaturation studies indicate that H420F may be more stable than wild-type tailspike. However, thermal denaturation suggests it may be less stable. Preliminary in vitro refolding studies indicate it may not form trimer at neutral pH. Additional experimentation is underway to help clarify the folding and stability of H420F.

 
 

Sensitivity Characterization of the Red Blood Cell Model Ryan Altenbaugh, K. J. Kauffman,  and Jeremy S. Edwards Department of Chemical Engineering Systems engineering tools provide insights into the multivariate regulation and pooling of complex biological networks that cannot be fully interpreted through experiments alone. We analyze the use of phase planes, temporal decomposition, and statistical analysis of the human red blood cell to explore the utility of each of these techniques for understanding the effect  of single parameter changes on the overall observed behavior of the network.  Specifically, several parameters in key regulatory steps in glycolysis, the Rapoport-Leubering Bypass, the pentose phosphate pathway, adenosine metabolism, and membrane transport were changed. The most sensitive parameters were identified based on the steady-state metabolite concentration changes and were explored further. Temporal decomposition identified time scale changes resulting from the parameter changes. These time scales were explored using phase plane and statistical analyses. Phase plane and statistical analyses both inferred changes in poolings, while statistical analysis also identified changes in the regulatory network structure that resulted from various parameter changes. Each of the system methods has strengths and weaknesses in exploring and gaining insight into complex biological networks.

 
 

Design of Genetically Engineered Proteins for Biomedical Applications Theresa Beinke1 and Kristi Kiick2,  Departments of 1Chemical Engineering and  2Materials Science and Engineering The cell binding domains of fibronectin contain two short peptide sequences, RGD and PHSRN, which are known to promote cell adhesion; in crystallographic studies these sites have been determined to be 30-40 Å apart. Multivalent displays of the RGD sequence have demonstrated improved binding to cellular targets over the free RGD peptide; including (PHSRN) in the displays in combination with RGD further increases binding affinity. In previous studies on polymers containing these peptide sequences, however, the peptides have not been placed in any controlled manner along the polymer chain. With the goal of creating polymeric molecules with improved cell binding, repetitive proteins have been engineered with regular placement of lysine and glutamic acid residues. After synthesis of the protein polymer, these groups will be modified with appropriate peptides, and their binding affinity measured. To produce the protein polymers, standard molecular biology protocols are employed. Seamless cloning strategies are being utilized, as this method permits multimerization of the repetitive protein without a sequence context and also allows short amino acid sequences to be appended at the N- and C-termini. The studies described here are a first step toward producing polymeric molecules to present the RGD and PHSRN sequences at varied distances to study the impact on cell binding. These artificial proteins may have important implications for biomedical engineering and material science because the control of polymer sequence may improve the interactions of the protein polymer with various biological targets.

 
 

An Optical Trap Microrheometer to Characterize Protein-Polymer Drug Delivery Networks John Bishop and Eric M. Furst, Department of Chemical Engineering In this project, we use optical trap microrheology to characterize the rheological properties of novel protein-polymer drug delivery networks. Microrheology enables us to measure the bulk viscoelastic moduli, local heterogeneity, and transient behavior of the protein-polymer networks, which determines the network’s release kinetics and erosion properties. The ability of optical tweezers to manipulate micron-scale particles precisely and non-destructively has formed the basis for a variety of applications in the biological and physical sciences. Another distinct experimental advantage of using optical tweezers is that extraordinarily small sample volumes, typically less than 10-20 microliters, are required. Experimentally, we have developed an optical trap rheometer that uses a function generator to drive a trapped colloidal particle in a sinusoidal motion, while simultaneously measuring the response of the particle with single particle tracking. Since the particle experiences fluid resistance when in motion, measuring the magnitude and phase of the particle displacement allows us to calculate the local viscoelastic storage and loss moduli.  The work done thus far has focused on programming a high-speed application to acquire an image, locate the particle’s position, and input the signal from the function generator at a rate of up to 30 frames per second. Preliminary results using polystyrene colloidal particles in water have been successful at characterizing the optical trap stiffness.

 
 

Novel Microfluidic Separations Using Optical Trapping Rich Dombrowski and Eric Furst Department of Chemical Engineering The objective of this study is to explore novel means of separating particles in microfluidic channels.  The separation strategy used in this project will employ arrays of optical traps to hold particles and divert them to other channels or selectively slow their passage.  Flow through a 2-dimensional array of traps will be investigated first in order to characterize the behavior of the system.  This study will also explore the effects of spatially or temporally varying the trapping strength on the selectivity of the separation.  Flows will be observed and analyzed using video microscopy and a computer framegrabber to track particles. The goal of this project is to develop monolithic devices that incorporate the optical trapping techniques along with analytical components for use as cell-sorters, immunoassays or DNA sequencing platforms.

 
 

Effects of Sulfate on Dechlorinatation and Methanogenesis in a Mixed Culture Andrew Joslyn and  Pei C. Chiu Department of Civil and Environmental Engineering Trichloroethene (TCE) and perchloroethene (PCE) are hazardous pollutants often found in water samples.  Microorganisms that can reductively transform these pollutants into nontoxic ethene have been discovered in the U.S. and abroad.  These bacteria play a key role in hazardous waste site remediation.  A TCE-dechlorinating anaerobic culture derived from Dover Air Force Base has been enriched in our laboratory and is known to contain Dehalococcoides ethenogenes, a bacterium that can dechlorinate TCE to ethene.  However, the interactions between this and other organisms in the culture are unclear.  In this study, the relationship between sulfate-reducing bacteria (SRB), homoacetogens, methanogens, and Dehalococcoides was examined by testing the effects of sulfate on TCE dechlorination and methane production.  Two electron donor-free media, one with sulfate and one without, were used to set up duplicate batch reactors.  Stock culture was centrifuged to obtain biomass, which was then re-suspended in fresh media and placed in 250-mL amber bottles in an anaerobic glove bag.  At different elapsed times, headspace samples (100 mL) were analyzed using a GC-FID that had been previously calibrated for this experiment.  Reactors containing sulfate showed 1,1- dichloroethene (1,1-DCE) as a major intermediate in the dechlorination of TCE to ethene, whereas sulfate-free bottles produced mostly cis-DCE as an intermediate.  Since 1,1-DCE is usually a minor intermediate during TCE reduction by Dehalococcoides, our preliminary result suggests that an organism other than Dehalococcoides was responsible for some of the TCE dechloination in the sulfate medium.  Additional testing will be conducted to determine if SRB were involved in 1,1-DCE production.

 
 

Delivery of Polymers to Plant Cell Walls Cory C. Mulcahy1, Timothy E. Proseus2, Norman J. Wagner1 and John S. Boyer2 1Department of Chemical Engineering, University of Delaware, Newark DE 2Department of Marine Science, University of Delaware, Lewes, DE Growth is necessary for all living things.  In this experiment we are looking specifically at the growth process in the cell walls of plants.  The alga Chara corallina was chosen for its large cell size which makes it simple to use.  Polymeric pectin and cellulose-building materials must be delivered to the existing wall for sustained growth.  Natural internal pressure (turgor pressure) extends the existing wall structure, and new wall material must be incorporated simultaneously.  In order to investigate the incorporation of the natural polymers. fluorescent molecules were pushed into isolated cell walls in front of pressurized mineral oil.  The mineral oil did not penetrate the wall, which remained in water.  Measurements of the fluorescence accumulated in a water bath surrounding the isolated walls were made as a function of time.  Initially, we used fluorescein isothiocyanate (FITC).  The data for the FITC leaving the cell were fit to existing diffusion models.  Using these models the effective diffusion coefficient was 4.63*10-11cm2/s, which is much smaller than 3.68*10-6cm2/s the value for FITC diffusing in water,.  Therefore the wall markedly decreases the movement of FITC.   Preliminary data suggest FITC is trapped or bound in water filled pores of the wall.  This process was repeated with FITC-Dextran molecules of a range of molecular weights.  The data show strong molecular size dependence and a pressure dependence for certain sizes of Dextran, but not for the much smaller FITC molecule itself.  Therefore, both the turgor pressure of the cell and the molecular size of the penetrating polymer control the introduction of material to the cell wall, and ultimately the growth within the cell wall.

 
 

Tracer Particle Microrheology of Actin Networks Melissa Plummer, Jennifer O’Donnell, and Eric Furst  Department of Chemical Engineering Actin is an important component of the cytoskeleton, and plays a critical role in cellular resistance, generation of stress, signaling and morphology. Understanding the rheology of actin will enable us to determine mechanical properties of cells, which will be useful for identifying and studying those with anomalous behavior, such as metastasized cancer cells.  Using tracer particles, we measure the high-frequency rheology of reconstituted filamentous actin networks (F-actin) with diffusing wave spectroscopy (DWS).  This was done by dispersing 0.992 µm and 2.834 µm polystyrene particles in F-actin, and then measuring the mean square displacement <delta r2(t)> of the particles with time. The polystyrene particles we use are uncoated, coated with BSA (bovine serum albumen), or coated with polylysine.  Probe particle Brownian motion reveals the total resistance to deformation and whether the resistance is viscous or elastic with the rate of strain.  We find that coating particles reduces the coupling to the actin network.  DWS microrheology indicates that the probe microenvironment is characteristic of polymer depletion from the particle surface.

 
 

Optimizing Expression and Gaining Insight into the Trafficking of Membrane Proteins Amy VanFossen, Ronald Niebauer, and Anne Skaja Robinson Department of Chemical Engineering The G-protein coupled receptors (GPCRs) are proteins that reside in the plasma membrane and deal with regulating a cell’s response to signals or molecules.  GPCRs are believed to play a role in heart disease and cancer so research about these proteins could lead to treatment of these conditions. One of the goals of the research is to produce large amounts of functional protein so detailed structural information of the proteins can be obtained. In order to track the movement of GPCRs, a green fluorescent protein (GFP) has been genetically fused to the protein as a label to monitor protein trafficking through the cells. For the substance P receptor (SPR), the protein does not make it to the plasma membrane.  Experiments show that SPR with the GFP label was fluorescing in the cell even though the SPR was not reaching the plasma membrane. Future work with the SPR involves unraveling why the protein is stopping on its way to the plasma membrane.  Another protein studied, the adenosine receptor (A2a), does make it to the plasma membrane thus the goal of all work for this protein is to produce the protein in high quantities. Currently, experiments concerning media and growth conditions are being done with A2a to optimize protein expression.

 
 

Investigating Mixed Chain Kinetics  of P22 Tailspike Dana Ungerbuehler and Anne Skaja Robinson,  Department of Chemical Engineering

Misfolded and aggregated proteins are implicated in several disorders such as Alzheimer’s, Cystic fibrosis, and prion diseases.  The lack of productive folding is thought to play a major role in the progression of these diseases, and some treatments are being sought in minimizing the tendency towards misfolding or maximizing the efficiency of these proteins.  The kinetics of protein folding is an area of great interest in biochemical engineering and pharmaceutical industries, and are extremely complex to model in vivo.  P22 tailspike is a protein-folding model due to its complex molecular interactions and oligomeric structure.  While the native trimeric structure is not disulfide bonded, evidence exists for a folding intermediate with oxidized sulfhydryl groups.  A C-terminal truncation revealed that the cysteines at 496, 613, and 635 were the most likely sites of sulfhydryl activity.  Cysteine to serine point mutations at the 496, 613, and 635 residues were purified in trimer form.  Initial analysis of the single serine mutants revealed that they folded 2-3 times slower than wild type and with only 65-80% of wild type yields.  In vitro refolding studies were performed by mixing different single mutants to investigate if wild type yield and assembly kinetics could be recovered.  Combining mutant chains has shown qualitative improvement in the yields, however the kinetics of folding appears to remain unchanged.  Thus, allowing mixed chain interactions do not produce more rapid folding, yet may produce more efficient folding.  More research will be conducted to characterize these interactions and investigate more favorable conditions for productive folding.

 
 

Effects of the R563K Mutation on P22 Tailspike’s Folding Jennifer Zak, Matthew J. Gage, and Anne Skaja Robinson Department of Chemical Engineering Improper protein folding is a critical factor in the development of various human diseases such as Alzheimer’s1 and some types of cancer2. The tendency for proteins associated with these diseases to aggregrate and become insoluble has been a major obstacle for research on these proteins. The tailspike protein from bacteriophage P22 serves as a good model protein to study protein folding because its folding and aggregation pathways are both well-characterized.  In addition, P22 tailspike forms a trimer, allowing the study of both folding and assembly processes at the same time.  This work has studied the role of amino acid 563 on proper folding and the stability of P22 tailspike.  Our hypothesis is that this amino acid is involved in a critical salt linkage that enables the trimer to assemble.   Arginine 563 was mutated to lysine (R563K) using site-directed mutagenesis. This changes the charge, and could disrupt any electrostatic interactions. R563K expresses as both  monomer and trimer in E. coli cells, indicating that this amino acid play a critical role in trimer assembly. Trimer from R563K expression was purified using anion-exchange and hydrophobic affinity chromatography. Refolding experiments were performed for the R563K mutation and for wild-type protein.  The refolding experiments resulted in a mixed population for the mutation (both trimer and monomer were present).  Further experimentation will attempt to determine the role of this amino acid site in the assembly of P22 tailspike trimer.  1Tjernberg, L. O., Callaway, D. J. E., Tjerberg, A., Hahne, S., Lilliehook, C., Terenius, L., Thyberg, J., and Nodstedt, C. (1999) JBC 274, 12619-12625. 2Lim, J. K., Lacy, M. Q., Kurtin, P. J., Kyle, R. A., and Gertz, M. A. (2001)  J. Clin Pathol 54, 642-646