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143 Cards in this Set
- Front
- Back
What's the Central Dogma of Molecular Biology?
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DNA-->RNA-->protein
(transcription) (translation) |
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Total # of genes estimated at ___________.
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35,000
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Genome is the ____________ component of an organism, while the Proteome is the __________ component.
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informational
functional |
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How many more proteins than genes?
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3x more proteins than genes
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sequence of bases is _______% the same in all people
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99.9%
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_____% of the genome codes for the production of proteins.
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less than 2%
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repeated sequences that do not code for proteins (junk DNA) makes up _____% of the genome
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at least 50%
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Name 3 methods of achieving diversity in the genome (and give examples for each).
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1) mRNA editing (ApoB)
2) splicing (alcohol dehydrogenase) 3) post-translational modification |
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___________ of the same enzyme perform different kinetics of the same basic function. These are generated by _________________.
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isoforms
alternate mRNA splicing |
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What causes 15% of all genetic diseases? Give example.
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defects in mRNA splicing
B-thalassemia |
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What are the levels of protein structure?
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1. primary (aa sequence)
2. secondary (alpha helix, beta sheet) 3. tertiary (fully folded peptide) 4. quaternary (homo/hetero subunit interactions or holoenzyme) 5. multiprotein machines |
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Give an example of a multi-protein machine. Tell how it works.
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pyruvate dehydrogenase (PDH)
changes pyruvate (from glycolysis) to acetyl CoA for the TCA cycle PDH is made of 60 polypeptides (5000 kDa!) |
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Proteins can function as enzyme. Give example.
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DNA polymerase
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Give example of protein functioning as structural.
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elastin
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Give example of toxin protein.
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Botox
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Give example of protein functioning as receptor.
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insulin receptor
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Give example of protein functioning as contractile.
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myosin
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Give example of protein functioning as a carrier.
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hemoglobin
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Give example of protein functioning as protective.
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Ig
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Give example of protein functioning as a ligand.
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insulin
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Give example of protein functioning as a transporter.
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ATP pump, cisplatin for Ca
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Which protein copies DNA prior to cell division? What type of protein is it?
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DNA polymerase
enzyme |
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How does Botox work?
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2 polypeptide chains
light chain protease cleaves SNARE complex, preventing release of ACh vesicles. heavy chain binds to cholinergic cell surface glycoproteins |
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Which muscles is Botox usually injected into to prevent formation of ___________ lines?
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corrugator muscles and procerus muscle
glabellar lines |
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What's a common cancer therapeutic? How can it be neutralized?
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DNA CROSSLINKING by CISPLATIN
(kills cells that replicate too rapidly) can be neutralized by glutathione and transporter pump |
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The level of a protein is determined by its rate of both ___________ and __________.
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synthesis and degradation
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DNA polymerase functions as an _________.
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enzyme
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How does B-thalassemia occur?
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pre-mRNA splicing defects
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Explain how the multidrug resistance protein works in regards to cancer chemotherapeutics.
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Cisplatin causes DNA crosslinks, preventing cancer cells from replicating.
The multidrug resistance comes from the transporter pump using glutathione to take cisplatin out. |
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What's the orientation of DNA synthesis?
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5' to 3'
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With bioinformatics, what is still the last step?
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confirmation by experimentation
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Proteins only contain the ___ isomer of amino acids.
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L
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What does Zwitterionic mean?
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Within the molecule, there are + and - charges, but the net is neutral at pH 7.
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Describe the amino acid. Which one is the simplest aa?
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alpha carbon
hydrogen amino group (NH3+) carboxyl group (COO-) variable R group glycine: R group is H |
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Name the non-polar amino acids.
What's another name for non-polar? |
hydrophobic
GLYCINE ALANINE VALINE (branched isopropyl) |
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Name the polar amino acids.
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HAVE -OH (targets for phosphorylation and glycosylation)
SERINE THREONINE |
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Name the basic amino acids. Which charge do they have at pH 7?
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+
LYSINE ARGININE |
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Name the acidic amino acids. What charge do they have at pH 7?
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-
ASPARTATE GLUTAMATE |
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Name the uncharged polar amino acids. What's another name for them?
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carboxiamide amino acids
ASPARAGINE GLUTAMINE |
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Explain the effect of pH on the ionization of an unsubstituted amino acid.
What happens if you lower/raise pH? |
isoelectric point is pH 7 (Zwitterionic form)
lower pH, protonate COOH, so now the aa is + raise pH, deprotonate NH2, so now the aa is - |
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In terms of pKa values, which groups are "neutral," acting as acid/base catalysts?
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histidine and cysteine
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In terms of pKa values, which aa's are acidic? Are these pKa values high or low?
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LOW (the aa's are - charged)
ASPARTIC ACID (ASPARTATE) GLUTAMIC ACID (GLUTAMATE) |
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In terms of pKa values, which aa's are basic? Are these pKa values high or low?
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HIGH (the aa's are + charged except for tyr)
TYROSINE LYSINE ARGININE |
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Given a polypeptide has 4 lysine residues, what is its charge at pH 7?
at pH 12? |
(lysine is basic, +, has high pKa)
so at pH 7 (neutral), it's POSITIVE (basic) at pH 12, you're deprotonating, making it more Neutral. |
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Name the acidic amino acids.
What's the charge at neutral pH? |
aspartate
glutamate - |
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Name the basic amino acids.
What's the charge at neutral pH? |
tyrosine
lysine arginine + |
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Which amino acid is really an imino acid?
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proline (has a ring)
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How does penicillin work?
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inhibits synthesis of bacterial cell wall peptidoglycan at the active site SERINE
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Tell me about cysteine residues. What molecule do they help stabilize?
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SH groups oxidize to S-S making inter/intramolecular disulfide covalent bonds.
insulin (polypeptide hormone) |
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What's the 21st amino acid? What's it's codon?
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selenocysteine (sec)
UGA (usually a stop codon) acts as an acidic aa b/c has low pKa |
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How can you use aa's as indicators of disease?
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look at serum levels as compared to normal ranges
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Low tyrosine levels are indicative of what disease?
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Phenylketonuria (PKU)
phenylalanine to tyrosine by phenylalanine hydroxylase. These kids have autosomal recessive deficiency in phenylalanine hydroxylase. Preventable by LOW PHE DIET. |
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What's a peptide bond? How do you break it?
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alpha carboxyl & alpha amino
(amide bond) It's how you link aa's. Requires energy. Water hydrolyzes the bond to give you free aa's again. |
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What's the favored stereochemistry of the peptide bond?
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trans. less steric hinderence.
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Which aa is basic, aromatic, and polar?
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tyrosine
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_______ bonds are rigid and do not rotate.
_______ bonds can rotate the rigid amide planes. |
peptide rigid
phi and psy bonds can rotate |
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AAs are always written ______ terminus to _______ terminus.
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+H3N to COO-
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What is a D aa analog that acts as an antibiotic? How?
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D-cycloserine
In bacterial, 5 gly residues are added to D-ala by glycopeptide tranpeptidase= cell wall. (inhibited by penicillin) Cycloserine is D-ala analog, so it acts as antibiotic. |
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Valine is _______ aa.
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non-polar
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Serine is _______ aa.
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polar (-OH)
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Tryptophan is ______ aa.
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aromatic
(b/c turkey smells good, haha) |
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Lysine is ______ aa.
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basic
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Aspartate is ________ aa.
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acidic
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Cysteine has a _____ group on it.
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thiol (sulphur)
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What has protein and RNA components?
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ribosomes
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What is the signal for import into the ER lumen?
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N-basic-hydrophobic-polar-C
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Which form of ribonuclease is active? Why?
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native with S-S b/c the denatured polypeptide has SH groups. The biological activity depends on the 3D structure.
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What dictates conformation of protein?
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primary sequence
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What happens in spongiform encephalopathies? Give 2 examples of these diseases.
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prion diseases, conformational change from alpha helices to beta sheets. Proteins aggregate.
examples: Creutzfeld-Jacob, Mad Cow |
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How do distant active site residues get close to each other?
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folding
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What are 3 ways to attack the bacterial cell wall?
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1) lysozyme cleaves NAM-NAG
2) penicillin blocks glycopeptide transpeptidase 3) D-cycloserine (seromycin) acts as a D-ala analog to gly residues don't attach |
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What's a zymogen?
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inactive precursors for proteolytic enzymes
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How are zymogens and insulin activated?
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proteolytic processing
-chymotrypsinogen (inactive in pancreas) -trypsin cleaves it, making 2 S-S bonds -autocatalyzes to alpha chymotrypsin (active) with 3 chains |
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How are HIV gp120 and glycogenin modified?
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glycolysis
(add sugar residues) |
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What are the 2 types of glycosylation and on which aa's does each type happen?
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1) N-linked (asn-asparagine)
2) O-linked (serine, threonine, tyrosine) |
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What's the functional structure of collagen?
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quaternary structure
ALPHA CHAIN TRIPLE HELIX (NOT alpha helix) |
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What's the alpha chain of collagen made of? (sequence)
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Glycine-proline-hydroxyproline
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What's a post-transl modification that's the suicide mechanism for methyltransferases?
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methylation of active site Cys (so now G=C doesn't base pair pair)
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What's the post-transl mod of histones to control chromatin structure? On which residues does this happen?
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acetylation
lysine residues decrease affinity of histone for DNA |
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What's the post-transl mod for insertion of proteins into membranes? Which residues are a target? Give an example.
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palmitoylation/myristylation
cysteine residues S-Palmitoylcysteine gets palmitic acid at the thioester. Anchors it into membrane by attachment of lipid groups. |
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How does apo enzyme differ from holo enzyme? What's an example?
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2 alpha subunits, 2 beta subunits
Hemoglobin with heme bound apo globin without heme bound. So they require non-proteinaceous groups bound for function. EXAMPLE: BIOTIN IN PYRUVATE CARBOXYLASE is the prosthetic group |
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Proteins are targeted to organelle locations by signals in their ____ sequence.
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primary
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_____________ directs proteins to their ultimate destination. How?
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Glycosylation.
In ER, mannose residues are added, phosphorylated in Golgi, the M6P residue targets to lysosome. |
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What's the targeting sequence for lysosomes?
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M6P (mannose 6 phosphate)
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What's the targeting sequence for mitochondria? Give an example.
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positively charged amphipathic alpha helix
influenza virus PBI-F2 (kills immune cells to ensure virus survival) |
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As a protein crosses the membrane, that stretch of aa's are ___________. Therefore, we can do ____________ plots to predict trans-membrane regions of polypeptides.
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hydrophobic
hydropathy plots |
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What's the molecular basis for prion disease?
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conformational change of normal protein causes aggregation
alpha to beta chaperones (GroES-GroEL) don't work |
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What does bioinformatics do? What's the last step always?
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-identify genes, protein coding sequences
-predict function, structure -trace evoluationary development -EXPERIMENTAL VERIFICATION |
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What are the 2 categories of homologs?
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1) orthologs (same structure & fcn, different species)
2) paralogs (same structure, different function) |
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What is active site conservation?
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different proteins with same catalytic functions
Both chymotrypsin and subtilisin have catalytic triad ser-his-asp at active sites. |
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What's a technique for primary structure determination?
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Edman Degradation (N-terminal aa sequencing)
use phenyl isothiocyanate to cleave PTH-N terminal aa. |
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What's a technique for secondary structure determination?
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circular dichroism
spectroscopic measurement of elipticity/absorbance of polarized light. (alpha helix high peak, beta sheet lower peak) |
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3 things about alpha helix:
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1) all R groups point out, H bonds at 1 and 4 parallel to axis
2) there are 3.6 residues per turn 3) there is a net dipole moment - --> + NEGATIVE TO POSITIVE (how optimistic!) |
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How do you determine the mass of a protein from the aa's?
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# aa's x 110 = mass of protein
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Give 2 examples of all alpha helical proteins
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hemoglobin and myoglobin
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What is a "helix breaker" ? Why? What is it found in?
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PROLINE b/c always steric hinderance
collagen alpha chains |
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2 things about Beta sheets
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1) R groups protrude above and below strand
2) H bonding of all C=O & N-H bonds |
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What causes reverse turns in beta sheets?
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proline
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Forces that define protein structure...
which are involved in hydroxylation of proline? |
covalent bonds
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forces that define protein structure...
which are involved in collagen triple helix? |
hydrogen bonding
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forces that define protein structure...
which are involved in leucine zipper? What drives the leucine zipper? |
hydrophobic interactions (dimerization with another leucine zipper - oligomerization)
increase in entropy (S) drives hydrophobic interactions (-delta G) |
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What is an acceptor in hydrogen bonds in proteins?
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water
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What's the weakest of the bonds?
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van der waals
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Many proteins ___________ into a quaternary structure.
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oligomerize
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Do enzymes work in isolation? What are they part of? Give an example.
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no.
multi-protein machines (pyruvate dehydrogenase) |
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What does gel filtration do?
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Separates based on size
(big stuff goes faster b/c small stuff enters beads) |
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What does ion exchange do? How?
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Separates based on charge
Based on % of charged aa's (basic: lys, acid: asp) (same chg goes thru faster b/c repelled) |
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What does protein electrophoresis do?
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separates based on MW and mass
little stuff goes faster. denatures proteins, reducing agent, makes them all - so they migrate to + |
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What's an ELISA?
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detects ANTIBODY in sample
Ag coat well, add serum to see if it has Ab, add enzyme-linked Ab, add color, color is proportional to amount of serum Ab. For HIV, must confirm with WB. |
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What does mass spectrometry do?
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gives exact mass of polypeptide
small ions fly faster thru field |
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What's another name for multi-enzyme machines?
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holoenzymes
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What's an example of a homodimer?
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DNA polymerase clamp (2 identical subunits)
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Tell the 4 post-trans mods done to collagen.
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1) hydroxylation of proline to hydroxyproline for more H bonding potential
2) lysine residues are hydroxylated, then glycosylated in Golgi 3) interstrand covalent crosslinks S-S at C terminus 4) collagenase cuts off globular ends --> fibril |
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What is osteogenesis imperfecta?
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mutations produce shortened collagen chains that "poison" triple helix stability
aa substitutions destabilize intrastrand bonding |
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Give an example of a multi-subunit enzyme.
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RNA polymerase (hetero-oligomerization of 4 different subunits)
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Give an example of a multifunctional enzyme.
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DNA polymerase III
(hetero-oligomerization of 10 subunits) alpha: DNA synthesis 5' to 3' beta: clamp gamma: clamp loader epsilon: 3' to 5' exonuclease |
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Give 2 examples of a multi-protein machine.
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20S proteosome
pyruvate dehydrogenase (60 polypeptides!) |
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What's Xeroderma Pigmentosum?
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Autosomal Recessive sensitivity to UV rays
defect in nucleotide excision repair mechanisms |
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How does emphysema occur in 2-5% of cases?
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mutations in alpha 1-antitrypsin, so elastin gets degraded by elastase.
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How does phosphorylation work? Which are the most often targeted aa's?
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serine, threonine, tyrosine (b/c have -OH)
terminal gamma phosphate gets used. Activates enzyme. Can remove PO4 with protein phosphatase to deactivate. |
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Name the aromatic amino acids.
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have polycyclic aromatic ring
PHENYLALANINE TRYPTOPHAN TYROSINE (has -OH) |
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What is the structure of influenza PBI-F2 protein? Where is it targeted to?
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+ charged alpha helical (arg and lys residues)
targeted to mitochondria |
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What's a technique for quaternary structure determination?
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genetic and biochemical screen to detect protein-protein interactions
x-ray diffraction and electron microscopy |
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The immunoglobulin (IgG) molecule is mostly _________ (structural). It has ______ chains, linked by _________.
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beta sheet
2 light chains, 2 heavy chains disulfide bond |
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Why do you need vitamin C to prevent scurvy?
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vitamin C (ascorbic acid) hydrolyzes proline to hydroxyproline, needed for the collagen chain stability
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What's the targeting sequence for nucleus?
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lysine residues
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In gluconeogenesis, what's required as a prosthetic group? How does it function?
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biotin
in pyruvate carboxylase, changes pyruvate to oxaloacetate (happens on lysine residues) |
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How do you break IgG into its subunits?
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proteolysis by papain breaks domains into Fab and Fc
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Insulin functions as a _________.
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ligand
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mRNA to RNA occurs in ___________. Process called ________.
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cytoplasm
translation |
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What's Maple Syrup Urine disease?
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patient makes too much valine
(hypervalinemia) |
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What's a Western Blot?
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detects presence of ANTIGEN in sample
gel, transfer, react w/ Ab, film, protein band |
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What degrades structural elastin? What degrades that? What disease does this lead to?
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ELASTASE
ALPHA1-ANTITRYPSIN (anti-elastase, prevents elastin degradation) Defects in alpha1-antitrypsin (or smoking) lead to EMPHYSEMA. |
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How do you get a protein degraded in a proteasome? What's a target for this on the polypeptide?
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Ubiquitination
(ubiquitin gets added at the C terminus) lysine residues are targets |
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How can you measure increases in protein purity?
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increase in specific activity
|
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What's a technique for tertiary structure determination?
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x-ray diffraction
(crystalize the protein) NMR |
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Which proteins cause a big multi-drug resistance problem? How do they work?
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transporters (ATP-dependent small molecule pumps)
Increase efflux of drug from the cell. |
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Proteins proceed through partially folded intermediates aided by _____________.
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chaperones
Hsp 70 (small) Hsp 60 (big) |
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What is a mixed beta sheet?
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parallel and anti-parallel
|
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Which aa's are targets for modification by phosphorylation/glycosylation? Why?
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polar aa's: serine & threonine
b/c have -OH groups |
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Which of these proteins would go to ER lumen?
a) N-tyr-glu-asp-C b) N-lys-val-ser-C c) N-trp-cys-glu-C d) N-ala-tyr-ser-C |
b) N-lys-val-ser-C
N-basic-hydrophobic (nonpolar)-polar-C |
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Making mRNA occurs in __________. Process is called ________.
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nucleus
transcription |
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Myosin is a ____________ protein.
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contractile
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