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69 Cards in this Set
- Front
- Back
the pKa
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pH at which half of the acid or base is dissociated
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Henderson-Hasselbach equation
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pH = pKa + log ([A-]/[HA])
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Amino Acid Residue convention for polypetides
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amino acid end is shown to the left and carboxyl end is drawn to the right
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primary
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level of protein structure consisting of the amino acid sequence and location of S-S bonds
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secondary
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consists of special repeating structures such as alpha helices and beta-pleated sheets
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Tertiary
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Total fold of a protein including repeating and unusual structures
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Quaternary
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the arrangement of different polypeptide chains in a protein complex
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prosthetic groups
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accessory components of proteins
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Differential centrifugation
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if a cell contains organelles, the first step of a purification might be this. Cell membranes will be centrifuged into a pellet at 1000 x gravity for 10 min and 20,000 x gravity at 20 minutes will yields a pellet with organelles
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solubility
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a cell extract containing soluble proteins is often fractionated by adding increasing concentrations of ammonium sulfate. Some proteins precipitate at rather low ammonium sulfate concentration while others require more.
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anion exchange chromatography
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chromatographic materials that contain cations such as DEAE as the stationary phase
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cation exchange chromatography
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chromatographic materials that contain anions such as phosphate and carboxymethyl as the stationary phases
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enzyme assay
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determines whether the proteins of interest is in the pellet or supernatant. Can use either a color assay or a radioactive tracer assay
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protein assay
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- Measures total protein by staining with the Bradford Reagent. The Bradford uses Coomassie brilliant blue dye which is brown at acid pH (max. absorption at 465 nm) and is blue when it binds to proteins (595 max. absorption)
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specific activity
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the number of enzyme activity units divided by the milligrams of protein
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SDS-PAGE
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sodium dodecyl sulfate polyacrylamide gel electrophoresis. SDS causes unfolding of the protein and makes proteins very negatively charged in rough proportion oto their mass.
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isoelectric focusing
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- the first step in a two dimensional gel electrophoresis which is a separation on the basis of isoelectric point. Each protein has positive and negative charges with different pKa’s and therefore has no charge at a specific pH, called the isoelectric point. Proteins are then separated on the basis of charge.
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Sanger reagent
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2,4-dinitrofluorobenzene reacts with amino groups
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Edman Degradation
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Amino terminal end of the protein reacts with phenyl isothiocyanate, releasing a phenylthiohydantoin derivative of N-terminal amino acid. The amino acid in this derivative can be identified and the procedure can be repeated for about 50 cycles. Cleavage is only 99% complete so the date become unclear after many embryos
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overlapping peptides
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used to help determine the entire sequence of protein when using edman degradation
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trypsin
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cleaves on the carboxyl side of positively charged amino acids
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chymotrypsin
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cleaves on the carboxyl side of aromatic amino acids
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cyanogen bromide
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cleaves on the carboxyl side of methionine
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locating s-s bonds
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one often finds S-S bridges between cysteine residues; make peptides by cleaving with trypsin for example on the native protein containing those bridges. The peptides can be seperated and then the separated peptides can be reduced by dithiothreitol to break the S-S ridges giving one cysteine in each peptide. Reaction of the reduced peptides with iodoacetate gives hydrogen iodide and carboxymethylated cysteines which are now stable and cannot reform S-S bonds. chomatography and electrophoresis on paper will separate two peptides that were linked, and their sequence analysis will indicate the location of the S-S bridges
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mass spectrometry
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used to determine an animo acid sequence by analyzing the fragments produced for molecular weight.
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Peptide synthesis
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carboxyl terminal amino acid is protected at its amino terminus with something called FMoc. The protected amino acid is reacted with resin to give linkage btw the resin and protected A.A. The protected A.A is deprotected by treatment with a mild organic base. The next amino acid is supplied in a form that is protected at its amino end by FMoc and activated at its carboxyl group by dicyclocarbodiimide. The second amino acid is added to the first and then deprotected with mild organic base. This addition can go on for many cycles. When the desired peptide has been made, it is released from the resin by treatment with hydrofluoric acid.
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x-ray diffraction analysis
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x-rays are passed through a crystal which are diffracted in specific ways that can be recorded on film. The nearer the atoms are to one another, the larger the angle of diffraction. This information can be used to work out the distribution of electrons, and eventually the structure of the molecule in the crystal
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Planar peptide bond
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The C-N bond of a peptide has double bonded character which means its rigid and planar
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trans configuration
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O and H have trans configuration in a peptide
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alpha helix
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a secondary structure of proteins; each amino acid takes up 1.5 A and there are 3/6 amino acids per turn. Can be a clockwise or counterclockwise helix.
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right-handed alpha helix
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an alpha helix that goes in a clockwise direction
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alpha keratin
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found in hair, wool, feathers, nails, and porcupine quills; keratin has a coiled coil which means it has two alpha helices coiled around one another
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structure of alpha-keratin
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two alpha helices wound around one another in a left handed coiled-coil and joined by S-S bonds
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Curling
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to curl hair, S-S bonds are reduced by a smelly S-H containing shampoo. Then, the hair is re-oxidized to stabilize the curly hair conformation.
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Helix breakers
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proline because it has no H on its amide N; amino acids 3 or 4 apart that are on the same side of the helix and are neighbors that are either ++ or --
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Helix makers
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amino acids 3 or 4 apart that are on the same side of the helix and are neighbors. Attraction (+) and (-) is a helix maker.
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B-pleated sheets
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common secondary structure; can be antiparallel or parallel in confirmation
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Anti-parallel B-pleated sheets
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C -> N and N -> C on reverse side; 3.5 A/amino acid in B-sheets. Spacing between is as follows: 3.5 for the glycine rows and 5.7 for alanine.
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Beta-turn
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3 or 4 amino acids per turn
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Beta-Alpha-Beta
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When a parallel B-sheet is joined by an alpha helix and forms a beta-alpha-beta moftif
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Beta-Barrel
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Beta-pleated sheet forms a barrel like structure; found in viral capsid proteins and in a bacterial enzyme that lyses red blood cells
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Collagen Helix
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Three left-handed helices joined in a right handed superhelix.
Structure of G X Y G: glycine X: often proline Y: Often 4-hydroxyproline Hydroxyprolines form H-bonds and make collagen stronger |
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Forces holding tertiary structure in place
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ionic bonds, H-bonds, van der waals interactions, the hydrophobic effect
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hydrophobic effect
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considerable clustering of aliphatic residues (valine, leucine, isoleucine, and phenylalanine) in the interior of the protein. It is like the separation of organic solvents and water. It is driven by an increase in molecular entropy
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S-S bonds
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disulfide bridges
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Ramachandran Plot
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plot of the allowed bond angles for amino acids in polypeptides. Only a minority of psi and phi angles are allowed.
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Denaturation
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when a protein becomes misfolded
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Renaturation
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When a protein folds back into its native conformation
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chaperonin
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a protein that helps some proteins fold. example of this is groE
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groE structure
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GroE consists of a small subunit and a large subunit. An unfolded protein has hydrophobic residues on its outside and these can associate with hydrophobic residues at the mouth of the GroEL cylinder.
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prion
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proteinaceous infectious only
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NMR
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nuclear magnetic resonance solves structures in solutions. utilizes the spins of 1H 13C and 15N to generate a magnetic dipole which align either parallel or antiparallel in a electric filed. A pulse cause some nuclei to reverse orientation. The absorption spectrum gives information about the nuclei and their environment
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Myoglobin
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protein that picks up oxygen in tissues
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Hemoglobin
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Blood protein that picks up oxygen in the lungs and deposits it in the tissues. Also picks up carbon dioxide in tissues and releases it into the lungs
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Heme
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porphyrin ring consisting of four linked pyrrole rings
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Porphyrin
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structure of heme
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Pyrrole
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coordinate a ferrous iron
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CO poisoning
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CO binds free heme irreversibly but at right angles to the heme group. To prevent this, the distal histidine of these protein changes the bonding angle between heme and CO so that CO's binding affinity is 100-fold reduced.
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hyperbolic binding curve
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myoglobin's binding curve is a hyperbola and is half-saturated at only .26 kPa
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sigmoidal binding curve
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hemoglobin's binding curve is sigmoidal (s-shaped) and is half saturated at 4 kPa
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hill coefficient
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measure of cooperativity; hemoglobin's hill coefficient is 2.8
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cooperativity
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in terms of hemoglobin allows subsequent oxygens to be able to bind better (ex. 1st doesn't bind that well but allows second to bind better and so on)
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2,3-bis-phosphoglycerate (BPG)
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BPG is needed for hemoglobin to dump oxygen to the tissues; at higher altitudes, BPG increases twofold over 36 hours.
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Bohr Effect
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Reducing the pH shifts the oxygen binding curve to the right sot hat oxygen is more easily lost in the tissues; same effect is seen when CO2 concentration is raised
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Allosteric transition
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change from one structure to the other
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tense state of hemoglobin
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deoxygenated form of hemoglobin
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relaxed state of hemoglobin
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oxygenated form of hemoglobin
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The effect of oxygen on the shape of hemoglobin
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binding of the first oxygen to one subunit bring the iron in hemoglobin into the plane of the of the porphyrin ring which draws the proximal hisdine toward the porphyrin ring shifting the position of the F-helix
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the effect of the shape of heme on the F-helix
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causes the contacts between the B2FG region and alpha1c helix to change dramatically
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