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- Creators: Johnston, Carol
- Creators: School of Molecular Sciences
Description
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine in various concentrations, and monitoring the output through UV-Vis spectroscopy. The reacted species was also analyzed via MALDI-TOF mass spectrometry. UV-Vis spectroscopic monitoring showed that in the presence of LAF-1 or tyrosine, the reaction between HRP and TMB occurred more quickly than the control, as well as in the highest concentration of LAF-1, the evolution of a peak at 482 nm. The analysis through MALDI-TOF spectrometry showed the development of a second peak likely due to the reaction between LAF-1 and TMB, as the Δ between the peaks is 229 Da and the size of the TMB species is 240 Da.
ContributorsDavis, Morgan (Author) / Ghirlanda, Giovanna (Thesis director) / Heyden, Matthias (Committee member) / Mazor, Yuval (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2022-12
Description
Objective: This research examined the impact of daily ingestions of commercial high protein nutrition bars (with or without added fiber) on 24-h energy intake and satiety
for one week among free-living young healthy adults.
Design: In a 4-week double-blind, randomized crossover trial, 21 normal and
overweight participants (Mean BMI 23.9 ± 2.7 kg/m²), free of chronic diseases, were
randomized assigned to HP (high protein: 21 g protein) or HPHF (high protein high fiber:
20g, 14 g fiber) nutrition bars. Participants were included in the trial if they meet the
criteria for non-smoking, and not taking prescribed medication for chronic diseases.
Participants were instructed to consume commercial nutrition bars daily for seven
consecutive days. Body composition was measured with a bioelectrical impedance scale
at weeks 1, 3, and 5. Dietary data was recorded by the MyFitnessPal app on Wednesday,
Friday, and Sunday of each week.
Results: The mean energy intake for the weeks HPHF bars were consumed is
significantly higher compared to baseline (1998 ± 534 vs. 1806 ± 537 respectively; p =
0.035). The mean fat mass following one week of HPHF bar consumption was
significantly higher than the baseline value (18.8 ± 6.8 vs. 18.3 ± 6.7 respectively; p =
0.023) and trended higher (18.8 ± 6.8 vs. 18.3 ± 6.7 respectively; p = 0.057) in
comparison to the value following one week of HP bar consumption. For the high
physical activity level group (n = 10), the mean energy intakes for the baseline week and
the weeks the HP and HPHF bars were consumed were 1883 ± 597 kcal, 2154 ± 712 kcal,
and 2099 ± 603 kcal respectively (p ˂ 0.04; energy intakes for both bars were
significantly different from baseline). Nutrient intakes differed significantly mirroring the
nutrient profile for each specific bar. There are significant effects after both bars on
satiety, but there were no differences between each bar.
Conclusions: Sales of nutrition bars gained rapid growth and may represent a
unique source for specific nutrients. However, ingestion of commercial high protein
nutrition bars may increase the risk of gaining fat mass and eventual body mass over
time.
ContributorsPang, Minghan (Author) / Johnston, Carol (Thesis advisor) / Shepard, Christina (Committee member) / Alexon, Christy (Committee member) / Arizona State University (Publisher)
Created2022
Description
Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) is responsible for the majority of carbon fixation and is also the least efficient enzyme on Earth. Rubisco assists 1,5-ribulose bisphosphate (RuBP) in binding CO2, however CO2 and oxygen have similar binding affinities to Rubisco, resulting in a low enzymatic efficiency. Rubisco activase (Rca) is an enzyme that removes inhibiting molecules from Rubisco’s active sites, promoting the Rubisco activity. The binding of Rubisco and Rca stimulates a high-rate of carbon fixation and lowers the overall CO2 concentration in the atmosphere. To study the interaction between the two complexes, Rubisco was extracted from baby spinach (Spinacia oleracea) and purified using anion-exchange chromatography and size-exclusion chromatography. Rca was designed to use a recombinant gene and overexpressed in Escherichia coli (E. coli). The purified proteins were verified using SDS-PAGE. The two proteins were assembled in vitro and the interaction of the protein complex was stabilized using glutaraldehyde cross-linking. The samples were then deposited on a carbon-coated electron microscopy (EM) grid, stained with uranyl formate, and observed under a transmission electron microscope (TEM). The ultimate goal is to image the specimen and reconstruct the structure of the protein complex at high resolution.
ContributorsHart, Hayden (Author) / Chiu, Po-Lin (Thesis director) / Redding, Kevin (Committee member) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Military Science (Contributor)
Created2022-05
Description
Plant-based eaters are known to reap nutritional benefits due to their dietchoice, but it is important to evaluate dietary differences that may put them at a disadvantage compared to omnivores. Stark differences exist in daily intakes of protein between vegans and omnivores, which may lead to several risks including decreased strength and bone density. The purpose of this study was to analyze the differences in protein intake, lean mass, strength, and bone density in vegans versus omnivores in order to support the argument for an increased recommended daily allowance (RDA) for protein for plant-based eaters. Participants in this study were assigned to groups based on omnivorous (n = 25) or vegan (n = 19) dietary pattern. Nineteen matched pairs were created based on age and BMI. Data was collected at a single lab visit and included health history and physical activity readiness questionnaires, 24-hr food recall, and anthropometric measures. Bone mineral density (BMD) was measured using DEXA and strength was assessed using hand and Biodex dynamometers. Statistical analyses were conducted using independent samples t-tests and Pearson’s correlation tests to evaluate differences in body composition, bone density, strength, and dietary intake between the two groups with significance set at p.05. Differences were seen in daily calorie (p=.007), protein (p<.001), fat (p<.001), and fiber (p=.009) intake. Lean mass (p=.282) and bone density (p=.651) were not different between groups, but lower body strength was different (p=.008). There was a correlation between lower body strength and protein intake (p<.001), and lean mass was correlated with lower body strength (p<.001), grip strength (p<.001), and bone density (p<.001), but not
i
LBM (p=0.158). Correlations were also observed between BMD and lower body strength (p=.004). These data suggest that there is a significant difference between protein intake in vegans versus omnivores, which appears to have a positive association with strength. BMD also has a positive association with strength as well as lean mass. Cumulatively, the results suggest that it may be beneficial for vegans to increase daily protein intake.
ContributorsNadalet, Camila R (Author) / Johnston, Carol (Thesis advisor) / Shepard, Christina (Committee member) / Hooshmand, Shirin (Committee member) / Arizona State University (Publisher)
Created2023
Description
Background: Vegan and vegetarian diets have gained in popularity in recent years. Stated reasons for this include some possible health benefits and concerns of animal welfare. Though considered to be nutritionally adequate, questions remain over whether current protein recommendations of 0.8 g/kg/d are sufficient to maintain body processes and growth. Protein is unique in that it is the only macronutrient that contains nitrogen. Its status can be determined through nitrogen balance analysis of the urine if protein content of the diet is known. Nitrogen balance is considered the gold standard for determining protein intake requirements. A negative balance indicates a catabolic state, whereas a positive nitrogen balance is seen during anabolism. In healthy people, nitrogen equilibrium is desired under normal circumstances. This equilibrium reflects the net synthesis and breakdown of proteins. While nitrogen balance techniques have been used for decades, currently, there are no known studies measuring nitrogen balance and protein intake in strict vegans. Methods: Twenty vegan, inactive, male participants were recruited and received a 5-day eucaloric diet with a known protein content held constant at 0.8 g/kg/d. On day five, 24-hour urine was collected by participants and aliquoted for future analysis. Nitrogen content of the urine was determined through photometric assay and compared to the known nitrogen content of the diet to calculate nitrogen balance status.
Results: Mean absolute nitrogen balance (-1.38 ± 1.22 g/d, effect size = -1.13) was significantly lower than zero (equilibrium) (p < .001). Mean relative nitrogen balance (-18.60 ± 16.96 mg/kg/d, effect size = -1.10) was significantly lower than zero (p < .001). There were no correlations seen between nitrogen balance and age, years as vegan, or fat- free mass.
Conclusion: Consuming 0.8 g/kg/d of protein is insufficient to produce nitrogen balance in long-term vegans.
ContributorsBartholomae, Eric (Author) / Johnston, Carol (Thesis advisor) / Sweazea, Karen (Committee member) / Wharton, Christopher (Committee member) / Lee, Chong (Committee member) / Kressler, Jochen (Committee member) / Arizona State University (Publisher)
Created2022
Description
Vegetarian diets can provide an abundance of nutrients when planned with care. However, research suggests that vegetarian diets may have lower protein quality than omnivore diets. Current protein recommendations assume that vegetarians obtain a majority of their protein from animal products, like dairy and eggs. Studies have shown that this assumption may not be valid. The recommended dietary allowance (RDA) may not be adequate in vegetarian populations with high protein requirements. The purpose of this study is to analyze dietary protein quality using the DIAAS (Digestible Indispensable Amino Acid Score) method in both vegetarian and omnivore endurance athletes. 38 omnivores and 22 vegetarians submitted 7-day food records which were assessed using nutrition analysis software (Food Processor, ESHA Research, Salem, OR, USA). Dietary intake data was used to calculate DIAAS and determine the amount of available dietary protein in subject diets. Dietary data was compared with the subjects’ lean body mass (obtained using DEXA scan technology), and strength (quantified using peak torque of leg extension and flexion using an isokinetic dynamometer). Statistical analyses revealed significantly higher available protein intake in the omnivore athletes (p<.001). There were significant correlations between available protein intake and strength (p=.016) and available protein intake and lean body mass (p<.001). Omnivore subjects had higher lean body mass than vegetarian subjects (p=.011). These results suggest that vegetarian athletes may benefit from higher overall protein intakes to make up for lower dietary protein quality.
ContributorsZuelke, Corinne (Author) / Johnston, Carol (Thesis advisor) / Wharton, Christopher (Christopher Mack), 1977- (Committee member) / Dixon, Kathleen (Committee member) / Arizona State University (Publisher)
Created2017