Term
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Definition
Polarity (ability to form H-bonds in solution)
Ability to ionize (act as weak acid/weak base in solution) |
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Term
| H-bonds are what type of interactions? |
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Definition
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Term
| Typical length of an O-H bond? Length of an H-bond in water? |
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Definition
O-H = 1 Angstrom
H-bond = 1.8 Angstroms |
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Term
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Definition
| Directional -> O---H-O bond is linear (forms a straight line) |
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Term
| In ice, one molecule of water forms how many neighboring H-bonds? |
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Definition
| Forms H-bonds with 4 neighbors (maximum) |
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Term
| Average lifetime of 1 H-bond? |
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Definition
| 10^-12 seconds = 1 picosecond |
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Term
| In liquid water, how many neighboring H-bonds & why? |
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Definition
| 3.4 bonds w/ neighbors; due to there being increased entropy in this state (less order) |
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Term
| Thermodynamic reasoning for the dissolution of glucose... |
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Definition
| It is a polar solute, so it can readily form H-bonds with water; as the solid disperses in the water, the entropy of the system increases (S>0); entropic effect of less of order of solute is main reason |
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Term
| Thermodynamic reasoning for dissolution of salt in water... |
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Definition
| Normally as a solid, see Na & Cl ions in crystal lattice structure; as it enters water, see electrostatic attraction of dipole in water molecules to the ions (6 moc H2O/ion) - causes entropy of the whole system to increase (freeing of ions from lattice) |
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Term
| Main interaction for NON-polar solutes in water? |
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Definition
| van der Waal's interactions - transient dipoles based on varying electron densities of atoms; NOT TRUE ELECTROSTATIC INTERACTIONS |
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Term
| Non-polar solute aggregation in water is known as... |
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Definition
| The Hydrophobic Effect; aggregation is mainly an ENTROPIC EFFECT (solutes aggregate to minimize SA exposed to water, so water becomes less ordered) |
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Term
| What is the primary contributor to protein conformational changes? |
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Definition
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Term
| Concentration of pure water |
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Definition
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Term
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Definition
| pH = pKa - 1 (90% HA, 10% A) |
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Term
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Definition
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Term
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Definition
| pH = pKa + 1 (10% HA, 90% A) |
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Term
| All but one aa is chiral, which is not? |
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Definition
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Term
| Which amino acid is NOT S (has R chirality)? |
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Definition
| Cysteine because of SH group |
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Term
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Definition
| The point at which a molecule has no net charge |
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Term
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Definition
R Mass = Amino Acid Mass - 18 (MW of water)
In a polypeptide, the N and C-terminal amino acids will be represented by amino acid MW and the internal aa's will be represented by residue masses |
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Term
| What is isoelectric focusing used for? |
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Definition
| To separate different proteins based on different pI values; to determine the unknown pI value of a protein |
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Term
| SDS-PAGE is what method of protein purification? |
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Definition
| Semi-quantitative method; determine the relative amounts of proteins in a sample (mole ratios) |
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Term
| What property does SDS-PAGE use to separate proteins? |
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Definition
| Separates proteins based on differences in MW (all proteins w/ SDS have same charge to mass ratio and roughly the same shape) |
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Term
| What is proportional to the amount of dye that binds to a protein? |
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Definition
| The protein's mass; heavier proteins bind more dye |
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Term
| What interactions typically hold oligomeric proteins together? How can we separate the individual protomers? |
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Definition
Hydrophobic interactions
Can use SDS to separate protomers - binds to hydrophobic surfaces which causes protomers to separate |
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Term
| What conditions are S-S bonds formed/broken in? |
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Definition
Formed = OXIDIZING
Broken = REDUCING |
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Term
| 2D Gel Electrophoresis... |
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Definition
| Combination of IEF and SDS PAGE; first use IEF to separate proteins based on pI, then SDS PAGE to separate based on MW |
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Term
| Where do aromatic aa side chains absorb mainly at? |
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Definition
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Term
| What are limitations of using A280 (absorbance) to measure the purity and amounts of a protein sample? |
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Definition
Not very sensitive (only to 50 micrograms/mL)
Can only use on random mixes; cannot be done on pure samples |
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Term
| If you have a pure sample of known F/Y/W composition, how can you calculate the concentration? |
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Definition
| Use the known relative amounts to determine the extinction coefficient -> then rearrange Beer's Law formula to calculate the concentration of protein |
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Term
| Can SDS break S-S bonds alone? |
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Definition
| NO. Need to add a reducing agent to break bonds |
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Term
| What interactions cause dye molecules to associate with proteins? |
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Definition
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Term
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Definition
| 1 unit = amount of protein it takes to convert 1 micromole of substrate in 1 minute at 25 degrees C |
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Term
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Definition
| Measure of purity; # of units/total protein (mg) |
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Term
| What is typically the 1st step in a purification process? |
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Definition
| Separation of proteins based on solubility differences |
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Term
| Solubility at LOW [salt]... |
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Definition
| Charges on different side-chains cause electrostatic interactions between proteins and they aggregate and precipitate out of solution (LOW solubility); little hydrophobic effects because [salt] is low |
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Term
| Solubility as [salt] increases... |
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Definition
| As the [salt] increases, the salt ions coat the proteins in "compensating charges" to reduce interactions (shields proteins) - less aggregation (HIGH solubility) |
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Term
| Solubility at HIGH [salt]... |
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Definition
At very high [salt] there are so many ions that they shield water molecules as well, interrupting with the H-bonding and other intermolecular interactions between the water and protein; less H2O to solvate proteins
Hydrophobic surfaces of proteins aggregate together to cause ppt out of solution (LOW solubility) - aggregation due to hydrophobic effects |
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Term
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Definition
| Then the protein is NET POSITIVE - charges repel each other (HIGH solubility) |
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Term
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Definition
| Proteins aggregate due to hydrophobic effects because they are neutral (LOW solubility) |
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Term
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Definition
| Proteins are NET NEGATIVE; charges repel and proteins remain in solution (HIGH solubility) |
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Term
| Typical column molecule in CATION EXCHANGE? |
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Definition
| Carboxymethyl cellulose (-CH2-COO) |
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Term
| Why is the hydrophobic effect more prevalent at high [salt]? |
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Definition
| It is due to an excess of ions in the solution which tie up more water molecules; this prevents them from solvating the proteins which allows for more hydrophobic binding (higher affinity to binding) |
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Term
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Definition
| Eluted volume of proteins TOO LARGE for the pores in size exclusion chromotography; = volume outside of the beads in the column |
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Term
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Definition
| Eluted volume of proteins which traveled through the beads in size exclusion chromotography; = void volume + volume of pores of beads |
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Term
| What is the ratio of elution volume/void volume dependent on? |
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Definition
| The shape of the protein itself; rod-shaped proteins of identical MW to spherical proteins will elute FASTER in size exclusion columns because they behave as larger proteins (length of rod = diameter of "larger protein") |
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Term
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Definition
Phenylisothiocyanate (PITC); benzene ring-N=C=S
Reacts with ALL free amino groups (including those at N-terminus and on Lys) |
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Term
| What can be used to recognize the PTHaa removed by Edman degradation? |
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Definition
| HPLC (combination of cation exchange and hydrophobic chromotography) |
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Term
| What conditions is Edman degradation run under? |
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Definition
| ALKALINE - need alkaline conditions to make the amino group a good nucleophile |
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Term
| Limitations of Edman Degradation |
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Definition
PITC reacts twice with Lys side chains; however, only cleaves the peptide bond
Limitation of less than 100 aa's
Often there is NO free N-terminus |
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Term
| Cleavage Sites of Enzymes |
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Definition
Trypsin - after K, R
Chymotrypsin - after F, Y, W
Elastase - after G, A, S, V
V8 Protease - after D, E
Asp-N - before D, E
****Serine proteases do not cleave before (on N-terminus side of) Pro |
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Term
| Problems with A.A. Composition sequencing? |
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Definition
Destroys all Trp
Hydrolyzes all amide bonds present -> converts all Asn to Asp and all Gln to Glu |
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Term
| Qualifications for a "similar" sequence? |
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Definition
| Greater than/equal to 25% sequence identity |
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Term
| Divergent evolution will produce which form of homologue? |
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Definition
| Paralogue - two similar proteins with usually different functions within the same organism |
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Term
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Definition
| 2 similar proteins in different organisms with the same function |
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Term
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Definition
| Fraction of bound receptors ([PL]/[P] + [PL]) |
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Term
| How does a molecule of dye bind to a protein? |
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Definition
| Binds hydrophobically (hydrophobic effect); will bind independent of the aa composition |
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Term
| To use standards to determine MW, need 2 things: |
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Definition
Need void volume < elution volume
Plot graph of Ve/Vo vs. logMW |
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Term
| How to elute affinity chromotography column? |
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Definition
| Add ligand in solution in free form (attached to nothing); binds to protein in mobile phase and causes it to bind less w/ the column, elutes |
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Term
| When using a His-tag, what is used to elute the column? |
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Definition
| Add in free IMIDAZOLE; mirrors the His side chain so that it competes for binding with the metals on the column (Co and Ni) |
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Term
| What is used to recognize PTHaa's? |
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Definition
| Characteristic elution times in a column; NOT the wavelength of absorption |
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Term
| What is the byproduct of cleavage w/ CNBr? |
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Definition
| Homoserine lactone where the Met residue was located |
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Term
| What molecule can be used to block S-S bonds from forming? |
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Definition
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Term
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Definition
| Leu and Ile cannot be distinguished; both have the same molecular weight |
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