Chemical and Biomolecular Engineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2751
Browse
4 results
Search Results
Item Translocation of protein cargo into Candida albicans using cell-penetrating peptides(2020) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections caused by Candida albicans pose a serious threat to public health. The rising drug resistance towards azoles, the current first-line antifungal treatment, warrants novel approaches to designing and delivering new antifungal agents that target C. albicans cells. To increase the intracellular delivery of bioactive molecular cargo, we studied the use of cell-penetrating peptides (CPPs) as delivery vehicles. CPPs have been extensively used to deliver different cargoes into mammalian cells, but limited work has focused on delivery into fungal cells. To improve understanding of CPP-mediated delivery to C. albicans, we studied their ability to deliver green fluorescent protein (GFP) intracellularly. For our work, we chose the CPPs, MPG and Hst5, that have previously shown translocation into fungal cells without cargo and recombinantly produced these CPP fusions to GFP in Escherichia coli. We investigated the CPP-mediated translocation of GFP using flow cytometry. Fusion of GFP to MPG resulted in translocation into 40% of C. albicans cells, which was significantly higher than 13% cells that demonstrate translocation of GFP without a CPP. However, Hst5 did not translocate GFP into cells, with only 5% of cells exhibiting Hst5-GFP translocation. Our results demonstrate that MPG can deliver GFP, while Hst5 is not as promising. These results are consistent with molecular dynamics simulations that show MPG enters a model membrane preferentially with the N-terminal residues, whereas Hst5 fails to enter the membrane. Our results emphasize the potential of CPPs in delivering large cargo to C. albicans cells and the advantage of using both experiments and simulations to study the translocation of CPPs into C. albicans. To explore factors affecting translocation efficacy, we evaluated the aggregation of CPP-GFP fusion constructs. Using dynamic light scattering and interference scattering microscopy, our results identified aggregation of our fusions at high concentration as a possible limitation to translocation, motivating future studies of the causes of aggregation and its relationship to translocation efficiency. Our work has shown that CPPs can deliver large biomolecular cargo into fungal cells and has laid the foundation for further studies to design better CPPs and to explore mechanisms limiting translocation of CPPs into fungal pathogens.Item OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION FOR CELL-PENETRATING PEPTIDE FUSIONS TO PROTEIN CARGO(2017) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant production of cell-penetrating peptides (CPPs) as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions; thus, ways to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions for fusions of five CPPs (NPFSD, pVEC, SynB, histatin-5 and MPG) to the cargo proteins biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP) and green fluorescent protein GFP. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37 oC for 6 h and 10 h led 2 to the highest levels of expression for the different CPP-cargo constructs. The fusion of histatin-5 to GFP was purified, and its translocation into the fungal pathogen Candida albicans was studied. The purified protein translocated into the nearly 3% of C. albicans cells. These results provide the foundation for future studies to improve translocation of varied CPP-cargo fusions into C. albicans cells.Item Computational simulations on membranes and a transmembrane protein(2017) Zhuang, Xiaohong; Klauda, Jeffery B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)To accurately model the transmembrane proteins, accurate descriptions of its natural environment, i.e., lipids, are critical. The all-atom CHARMM36 lipid force field (C36FF-AA) is tested with molecular dynamics (MD) simulations. Through comparison to experiments, we conclude that the C36FF-AA is accurate for use with bilayers of varying head and chain types over biologically relevant temperatures. The united-atom chain model of the C36FF (C36FF-UA) of common lipids is developed to improve simulation efficiency. It shows good agreement between the simulated bilayer properties obtained by C36FF-UA and experiments, and also between the simulated results from UA and AA lipid models. Besides the single-component membrane, multiple-components 18:2 linoleoyl-containing soybean membrane models have been developed. The structural properties of pure linoleoyl bilayers agree well with experiments, based on which the soybean membrane models also result in reasonable structural properties. Accurate lipid force field greatly facilitates the study of transmembrane proteins. Lactose permease of Escherichia coli (E. coli) belongs to major facilitator superfamily (MFS) which is the largest and most diverse family of transporters and serves as a model for secondary active transporters (SATs) in this dissertation. LacY structures of the cytoplasmic-open, occluded-like, and recently periplasmic-partially-open state have been determined, however, the crystal structure of LacY in the periplasmic-open state is still not available. The periplasmic-open LacY structure is important for understanding the complete proton/sugar transport process of LacY as well as other similar SAT proteins. MD simulations are performed to test the accuracy of the previously developed periplasmic-open LacYIM-EX model (JMB 404:506), and two other periplasmic-open LacY models, LacYSW and LacYFP models (JMB 407:698). The simulated results indicate that LacYIM-EX is the only structure that remains stable in the periplasmic-open state. The MD dummy spin label simulations (MDDS) have also been performed and the results show that the orientation of the spin labels significantly affect the distance measurement so that the proper interpretation of DEER requires the aid of MDDS simulations. Self-guided Langevin dynamics (SGLD) simulations are performed to search periplasmic-open LacY. The results show that no outward-facing is obtained with nanosecond-averaged results, but if we study individual structures, conformational sampling is obtained with certain SGLD parameters that enhance natural helical motions. This SGLD approach might hold promise for studying conformational changes of other SAT proteins.Item TOBACCO MOSAIC VIRUS BASED THREE DIMENSIONAL ANODES FOR LITHIUM ION BATTERIES(2011) Chen, Xilin; Wang, Chunsheng; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Silicon and tin are promising anodic materials with both the high gravimetric and volumetric capacities for the next generation lithium-ion batteries. To prevent silicon or tin electrodes from a structure failure due to the volume change during lithiation and delithiation, a genetically modified Tobacco mosaic virus (TMV1cys) template is used to fabricate a 3D current collector for the silicon or tin electrode. The 3D current collector can effectively enhance the stabilities of the silicon or tin anodes. The TMV1cys particle can vertically self assemble onto the metal (i.e. Au, Ni, Fe) surfaces in a buffer solution ( PH=7 ). The abundant cysteine-derived thiol groups on the outer surface of the TMV1cys particle can react with metals to form near-covalent bonds. Thus it is very simple to form a 3D current collector by reducing metal such as nickel onto the TMV1cys surface by an electroless metal deposition. The 3D structure increases the electrode surface area by 10-fold. In order to investigate the effect of the 3D structure on the silicon anode, a physical vapor deposition methodology is used to deposit silicon onto the 3D current collector to form a nickel-silicon core-shell nano-rod anode. The abundant free spaces in the electrode accommodate the volume change during cycling and thus the cycleability of the silicon anode is greatly enhanced. The retention capacity at 1C is more than 1100 mAh/g after 340 cycles. Furthermore, a simple electrodeposition method is used to replace the complex physical vapor deposition methodology to make a uniform silicon deposition on the 3D current collector. The electrodeposition methodology is also used to prepare a tin anode. The electrodeposited silicon anode has comparable performance to those silicon anodes prepared by the physical vapor deposition technique. In order to enhance the electrochemical kinetics in silicon anode, the phosphorus doped n-type silicon is used to replace the pure silicon for preparing a high-rate-performance 3D silicon anode. Since the electrochemical reactions take place on the interface between the silicon and the electrolyte, the n-type silicon provides a quicker diffusion path for the involved electrons. The rate capability of the silicon anode has been increased and the capacity difference enlarges with the increasing current density.