Browsing by Author "Pathange, Lakshmi Prasad"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Characterization of protein microstructure by various chromatographic techniques
    Pathange, Lakshmi Prasad (Virginia Tech, 2007-02-28)
    Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters. The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface. PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC. The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media. For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values. In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments.
  • Thumbnail Image
    Non-Destructive Evaluation of Apple Maturity Using an Electronic Nose System
    Pathange, Lakshmi Prasad (Virginia Tech, 2003-02-06)
    The apple growers and packaging houses are interested in methods that can evaluate the quality of apples non-destructively. Harvested fruits are a mixture of immature, mature, and over mature fruits, thereby posing a great problem in deciding their end use and storage time. It is expected that the technique developed from the present project could be effectively used to classify the harvested fruit into immature, mature and over mature apples, rapidly and non-destructively. It would also help the growers to predict the optimum dates to harvest the fruits. York and Gala were the varieties of apples that were used in this study and were obtained from Virginia Tech College of Agriculture and Life Sciences Kentland Farm. Apples were harvested at different times resulting in different maturity groups (immature, mature and ripe). Gala apples were harvested on three dates with an interval of 10 days, while York apples were harvested on four dates with an interval of 14 days. They were stored at 0oC until sampled. For each harvest date, the experiments were conducted in two sets (10 each) on two consecutive days. First the ethylene levels were measured, followed by gas chromatograph and electronic nose. Then the maturity indices were measured. Three maturity indices, starch index, firmness and soluble solids were used as the three variables for the statistical analysis to identify and categorize the fruits into three maturity categories referred as immature, mature and over mature fruits. Apples were also categorized into three maturity groups based on the emanation levels of the aroma compounds evolved from the fruits. Then electronic nose sensor responses were categorized into the above maturity categories, and their effectiveness was determined using a statistical procedure called Discriminant Analysis (DA). From the DA cross validation results the correct classification percentage for Gala and York apples into maturity groups was 95%. The Electronic nose sensor's effectiveness to categorize the same observations based on sensor responses in to the above classified maturity categories was 83% correct in case Gala apples and 69% for York apples. The EN sensors response data were analyzed by the EN system software and the correct classification percentage for Gala was 83% and for York was 81%. Aroma-based categorization for Gala apples was 100% correct, while the electronic nose for the same analysis was 80%. Based on the three physical parameters, an objective evaluation of maturity could be accomplished. Principal Component Analysis, Canonical Discriminant Analysis and DA results demonstrated that the electronic nose could be used to classify apples into three identified maturity-based groups. The EN sensors (Gala apples), could also classify the apples into aroma-based categories. Thus, it can be concluded that the EN system holds promise as non-destructive evaluation technique to determine the maturity of an apple.