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72 Cards in this Set
- Front
- Back
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biologically relevant stereoisomer of amino acids
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L isomer (S stereoisomer)
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at pH 7, most amino acids are....
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zwitterionic; carboxyl group deprotonated, amine protonated
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Glycine
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Gly, G
simplest a.a. side chain=H |
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Alanine
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Ala, A
side chain=methyl group |
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hydrophobic amino acids
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Valine, Leucine, Isoleucine, Methionine
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Valine
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Val, V
hydrophobic side chain=isopropyl (3 C's) |
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Leucine
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Leu, L
hydrophobic side chain=4 C's (isopropyl end) |
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Isoleucine
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Ile, I
hydrophobic side chain=4 C's (sec butyl) |
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Mehthionine
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Met, M
hydrophobic side chain=CH2-CH2-S-CH3 (no branching) |
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2 special amino acids
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proline, cysteine
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Proline
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Pro, P
special a.a. side chain=cyclic (3C's bound to NH2) |
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Aromatic Amino Acids
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Phenylalanine, Tyrosine, Tryptophan
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Phenylalanine
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Phe, F
side chain=phenyl hydrophobic |
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Tyrosine (pka)
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Tyr, Y
hydrophobic except OH can participate in H-bonding side chain=phenyl group + OH (opposite from where it is attached) pKa around 10 |
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Tryptophan
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Trp, W
hydrophobic except amine can participate in H-bonding side chain=2 cyclic rings--5-membered with N and 6-membered |
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hydrophilic amino acids
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serine and threonine
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Serine
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Ser, S
hydrophilic side chain=CH2OH |
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Threonine
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Thr, T
hydrophilic side chain=CH with methyl and carboxyl group |
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Cysteine (pka)
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Cys, C
special amino acid because the sulfhydryl group (-SH) can oxidize to form disulfide bond with neighboring cysteine (termed Cystine) side chain=CH2SH 2 bonded together=R-S-S-R + H2O2 pka=8.3 |
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basic amino acids
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Lysine, Arginine, Histidine (positively charged at neutral pH)
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Lysine
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Lys, K
basic amino acid side chain=large, terminates in amine group (NH3+) pKa=10.5 |
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Arginine
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Arg, R
basic amino acid side chain=large, terminates in N-C+ bonded to 2 NH2 groups side chain pKa=12.4 (most basic) |
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Histidine
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His, H
basic amino acid side chain=large, terminates in 5-membered ring with 2 NH's pka=6 |
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carboxylate amino acids + derivatives
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Aspartate, Glutamate, Asparagine, Glutamine
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Aspartate
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Asp, D
carboxylate amino acid side chain=negatively charged CH2-C bonded to 2 O's (smaller than glutamate) pKa around 4 |
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Glutamate
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Glu, E
carboxylate amino acid side chain=ends in negatively charged C bonded to 2 O's pKa around 4 |
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Asparagine
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Asn, N (or aspartic acid, Asx, B)
derivative of carboxylate a.a. side chain=CH2-C double bonded to O and bonded to NH2 |
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Glutamine
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Gln, Q (or glutamic acid Glx, Z)
derivative of carboxylate a.a. side chain=ends in C double bonded to O and bonded to NH2 (larger than Asn) |
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protein sequences are numbered from what side to the other?
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numbered from N to carboxyl
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insulin A and B chains are linked by...
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disulfide bonds between cysteines
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Torsional angles
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Psi: Calpha-C bond
Phi: Calpha-N bond |
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most favorable orientation of Calpha bond angles can be identified on a...
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Ramachandran diagram
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alpha helix
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regularly repeating structure of amino acids
3.6 residues/turn, 5.4A/turn CO and NH groups are H-bonded r-handed helix all side chains are on outside O is referred to as i, H-N is referred to as i+4 |
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example of protein that is almost entirely alpha helical
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ferritin
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examples of alpha-helical coiled coil
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myosin, fibrin, keratin, intermediate filaments
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beta-sheet
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CO group of amino acid in one B-strand forms H-bond with NH group of amino acid in adjacent strand--parallel or antiparallel
side chains point in opposite directions B-sheets are pleated |
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B-sheets are depicted by...
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arrows N--->C
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beta-barrels
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B-sheets often fold back in on themselves to form beta-barrels
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loops in protein structures
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between B-sheets or a-helices
often important functional domains |
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globular proteins (how long)
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usually 100-400 amino acids long
hydrophobic parts fold inward |
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oligopeptide
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small protein
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2 denaturing agents
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urea and guanidinium chloride
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ribonuclease A
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4 pairs of disulfide bonds; not denatured in presence of denaturing agents (urea or guanidinium cl)--must be reduced by B-mercaptoethanol; once denatured, incorrect pairs of disulfide bonds can form; adding trace quantities of B-mercaptoethanol can cause correct SS bonds to form because that is the most energetically stable
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protein involved in BSE and other prion diseases
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PrP (PrPsc is infectious form)
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amyloid fibers
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contribute to neurodegenerative disorders
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3 fractions of centrifugation
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pellet 1: nuclear fraction
pellet 2: mitochondrial fraction pellet 3: microsomal fraction |
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salting out
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using ammonium sulfate at high concentrations--some proteins precipitate at 25% saturation, others at 50% saturation--use centrifugation to isolate precipitated proteins in native conformations
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dialysis
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separation of proteins by size--dialysis membranes have pores of specified size--any molecule or protein smaller than the pore size diffuses through membrane and out of dialysis bag
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gel-filtration chromatography
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separation of proteins based on shape--use carbohydrate beads--small molecules can go through beads, so larger proteins elute faster; approximate molecular weight can be estimated from standard consisting of proteins of known weight
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PAGE
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polyacrylamide gel electrophoresis; molecules are separated by size and charge in electric field--smaller molecules migrate more rapidly through porous gel; separates proteins in native state
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SDS-PAGE
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sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS is a negatively charged detergent which denatures proteins, SS bonds are reduced by B-mercaptoethanol; proteins must be stained to see them--SDS-PAGE is done in unfolded state
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ionizable groups in proteins that are deprotonated at pH 7
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terminal alpha-carboxyl group (-1)
aspartic acid/glutamic acid (-1) ~histidine (0; pKa=6) |
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ionizable groups in proteins that are protonated at pH 7 (5)
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terminal alpha-amino group (+1)
cysteine (0) tyrosine (0) lysine (+1) arginine (+1) |
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ion exchange chromatography
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separation based on charge; positively charged protein binds to neg. charged beads; elute positively charged protein bound to negatively charged beads using gradient of increasing salt (competes with protein for binding to beads)
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types of ion exchange chromatogrphy media
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cellulose or agargose bound to carboxymethyl (ionized form)--binds to + charged basic residues
cellulose or agarose bound to dethylaminoethyl (protonated form)--binds to - charged acidic residues |
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isoelectric focusing
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proteins loaded onto gel with gradient of low-high pH values; voltage applied to gel; proteins migrate until they reach pH in which they have a net charge of (isoelectric point or pI value)
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isoelectric point
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pH at which net charge of a protein is 0
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2D gel electrophoresis
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1) separate proteins by isoelectric focusing
2) apply isoelectrically focused proteins to an SDS-PAGE gel and separate by mass |
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affinity chromatography
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small molecules are attached to beads--proteins that bind small molecule are retained in column; can be eluted with small competing molecule or with salt gradient; ver powerful--can yield >95% purity
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5 examples of affinity interactions
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-metal binding proteins
-ATP binding proteins -drug binding proteins -protein-protein interactions -antibody-protein interactions |
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Western Blotting
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SDS-PAGE, transfer to polymer sheet, add radiolabeled antibody then wash to remove unbound antibody, overlay photgraphic film, expose, develop (autoradiogram)
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high pressure liquid chromatogrphy
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high pressure limits diffusion, increases interactions with chromotography media; HPLC yields very high resolution purification of protein components;applicable to many chromotography techniques
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assessing protein purity
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can be assessed on SDS-PAGE gel
enzymes--specific activity of purified enzyme can be determined using assay and enzyme's concentration (units of activity/mg protein); i.e. lactate dehydrogenase (lactate-->pyruvate) |
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Edman degradataion
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proteins are N-terminally sequenced
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ways of protein sequencing
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Edman degradation
mass spectrometry DNA sequencing |
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mass spectrometry
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determines mass of proteins and peptides
1) sample is ionized 2) electrical field accelerates ions 3) lightest ions arrive at detector first 4) laser triggers clock |
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in mass spectrometry, time of flight is related to
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mass to charge ratio
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X-ray diffraction
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determines protein structure; x-ray beams into crystal, diffracted beams register on detector (e.g. film)
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NMR Spectroscopy
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measures change in energy--transition between spin states gives NMR line
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NOESY
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Nuclear Overhauser Enhancement Spectroscopy; 2D spectrum--shows pairs of protons in close proximity; 2 nuclei must be less than 5A apart in order to show up as close together; in aqueous state, so side chains can move some
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_______ conformation of peptide bond is favored for all amino acids except_______
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trans; proline
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pka of terminal amino group and carboxyl group
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carboxyl=3.1
amino=8 |
