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23 Cards in this Set
- Front
- Back
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Circular Dichroism
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differential absorption of circular polarized light rotating in opposite directions; secondary structure of proteins info
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MALDI-TOF
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determines molar masses and sequencing of repeat units by using a laser beam to soft ionize the protein, while using the matrix to protect it and facilitate ionization; uses mass m and charge z to travel distance (1/2)(m/z) to calculate ion mass
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SDS-PAGE
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separate the components of a protein by their length or molecular weight by running the protein through a gel; SDS treatment prior to running through the gel causes proteins to denature and dissociate into subunits, and therefore the chain length determines the migration rate in the polyacrilamide gel
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FNMR Spectroscopy
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analysis technique that provides a high res 3d structure of the protein
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HPLC
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a chromatography technique in which the components of a mixture are separated by dissolving in a solvent and then forcing it to flow through a chromatographic column under high pressure; different types of HPLC modes (reverse-phase, normal phase, ion-exchange chromatography) reveal different types of interactions between components
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hydrophobic collapse model
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folding is influenced by having nonpolar hydrophobic AA buried in the core of 3D structure, while charged polar regions are on protein surface
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nucleation-condensation mechanism
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hierarchical folding; structure of transition state is built around a nucleation site while the rest of the protein is in the process of collapsing around the nucleation site; follows two-state kinetics without accumulation of intermediates
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transition mutation
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a purine/pyramidine base pair is replaced with a base pair in the same purine/pyramidine relationship (e.g. GC with AT)
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transversion mutation
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a purine/pyramidine replaces a pyramidine/purine base pair or vice versa (e.g. GC with TA)
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# of variants with M mutations and N amino acids
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19^M [(N!/(N-M)!)*M!]
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how to test oligomerizational state of proteins?
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tested by analytical centrifugation experiments, which determines molecular weights and compares them with theoretical values
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S5 alkenyl amino acid
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increases helical content of peptides by stapling reaction catalyzed by ring-closing olefin metathesis
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Similarities & differences between bacterial collagen and animal collagen?
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Both have a non-triple helix region surrounding the triple helix domain.
Difference: bacterial collagen have amino acid trend repeats such as a specific amino acid rich region |
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Method used to express proteins
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1. Pick colony from streaked plate
2. Grow start colony by inoculating in media 3. Inoculate in fresh expression media 4. Induction by using appropriate inducer |
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Methods to improve protein yields
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a. Increase time/temp of induction & concentration of inducer
b. Use appropriate codon for corresponding host c. Increase aeration speed d. use protease deficient host to prevent proteolytic degradation e. Use lower growth temp in case of proteolytic degradation |
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expression vectors
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plasmids, lambda phage, cosmid and yeast artificial chromosome
Lambda phage has very high transformation efficiency (1000 times more than plasmid) Cosmids and YACs carry large amounts of DNA (45kb and 2 Mb) |
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PLP mediated transamination
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process involves N-terminal amino group transformed into a ketone or aldehyde with help of PLP to react with alkoxyamines containing functional groups of interest
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Catalytic promiscuity of PAS
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PAS has catalytic promiscuity on sulfate and phosphate monoester because both have similar bond lengths, geometries, and transition-state charge changes.
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Competitive Inhibition
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Both lines meet at the same point on the y-axis
Mechanism: binds in active site, competing with substrate |
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Non-competitive inhibition
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Inhibited line is higher up on y-axis; both lines meet at x-axis
Mechanism: Only small addition of inhibitor binds to enzyme |
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Uncompetitive inhibition
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Both lines are parallel
Mechanism: ES complex binds to inhibitor, not at all to free enzyme |
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Mixed inhibtion
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Inhibitor is higher up on y-axis and do NOT meet at x-axis
Mechanism: Free enzyme and ES complex bind to inhibitor at same time |
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negative design
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implies consideration of undesired end-points to a given start-point and the goal of avoiding these undesired products by tuning the start-points
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