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What are the components of amino acids (5)
- 1. Alpha carbon (chiral except in glycine [R=H])
- 2. Hydrogen
- 3. R group (gives AA its properties)
- 4. Amine group (NH3+ at neutral pH)
- 5. Carboxylate acid group (COO- at neutral pH)
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Hydrophobic R Groups
rich in hydrocarbons
hydrophobic interactions are on the INSIDE of a water-soluble protein
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Hydrophilic R Groups
have oxygens, nitrogens & ESPECIALLY charged groups
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Protein Backbone
linear chain formed by N & 2 C atoms
proteins adopt particular shapes to maximize favorable atomic interactions to make a shape with a function
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What forces dictate protein folding & stability?
the sum of weak, non-covalent (low energy) bonds [1-7 kcal/mol v. covalent forces: > 50 kcal/mol]
• some proteins don’t fold easily on their own & to do so require the assistance of chaperones
• destabilizing amino acid mutations disrupt fold(ing)
• unfolded proteins can aggregate & be improperly trafficked
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How can you predict the function of a protein who’s role is unknown?
by looking at its AA sequence & seeing if it’s homologous to other proteins’
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Specific Activity
the amount of activity per amount of purified protein
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How can one measure the amount of purified protein?
• insert a sample of protein in a spectrophotometer calibrated to detect absorbance of the sample at a wavelength of 280nm
• tryptophan & tyrosine absorb UV light at 280 nm
• amount of light produced ~ amount of protein?
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Alpha Helix
secondary protein structure held together via hydrogen bonds that occur between the oxygen of a carbonyl group & the HN of an amino acid that is 4 residues down the polypeptide chain
• protein with charged residues (eg. Lys Lys) adjacent to each other in sequence can’t exist in a helix because the shape of it puts them too near each other
• CAN be AMPHIPATHIC (having both hydrophilic & phobic parts)
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Which amino acid cannot fit in an alpha-helix?
Proline: has a five-member ring & is referred to as the “helix breaker”
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Beta Sheet
secondary protein structure held together via hydrogen bonds that occur between backbone atoms within stretches of amino acid residues
• at least 2 (usually more) extended polypeptide chains are involved
• prolines can exist in beta sheets
• adjacent R groups can be 180° apart so it’s a favorable conformation for AAs that have the same charge & exist next to each other in the peptide chain
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CFTR protein
transmembrane protein of the ATP Binding Cassette family with 5 domains
• activation allows passage of Cl - ions through the PM via 2 methods (phosphorylation of the R domain & ATP binding + hydrolysis by NBD domains)
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What are the 5 domains of the CFTR protein?
- • TM1, TM2: 2 transmembrane domains that are groups of 6 alpha helices that form a chloride channel
- - these helices are amphipathic: hydrophilic on the inside conducive to Cl- & hydrophobic on the outside conducive to placement in the PM
- • NBD1, NBD2: 2 cytoplasmic nucleotide binding domains that bind & hydrolyze ATP
- - a lot of mutations occur in the nucleotide binding domain
• R domain: a regulatory domain modified by phosphorylation
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Describe how CFTR is activated:
• R domain phosphorylation activates the channel
• ATP hydrolysis by NBD domains changes channel conformation & allows passage of Cl- ions through the PM
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Kalydeco
a treatment for CF patients with the G551D mutation
• fixes CFTR protein misfolding → allows it to fold properly
• only assists 4-5% of people with CF (75% have the deltaF508 mutation)
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Glycine
• has the smallest amino acid side chain (R = Hydrogen)
• allows for sharp bends in chains & for chains to come close to each other (eg. hairpin bend in p53 protein)
• can be hydrophilic or hydrophobic due to its minimal H side chain
• the CF G551 mutation is one that changes a Glycine to another AA → disrupting CFTR function
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Name 2 Fibrous Proteins:
1. Elastin
2. Keratin
are relatively insoluble in water & are ELONGATED instead of being compact
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Elastin
- fibrous protein found in elastic tissue (eg. Lungs)
- the major kind of protein found in elastic tissue
- has no regular 2ndary structure
- is relatively water insoluble & elongated
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Desmosine Residue
a polypeptide connection involving four Lysines found only in elastin (springy connective tissue protein)
[connects spaghetti noodles that make up elastin]
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Keratin
- tough fibers rich in Cysteine
- contains tightly wound alpha-helices that are difficult to digested
- protein of hair, nails & skin
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Hereditary Acidosis
• disease in which there is increased acidity in the blood & other body tissue
• can result in growth defects, eg. bone formation issues, cognitive problems
• treatment is often ingesting bicarbonate
• causes problems in protein function because if protein isn’t at the right pH, it’s side chains will have wrong charge & may not function
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pH
- the negative log of the hydrogen ion concentration
- when [H+]=10-7 M → pH = 7
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pKa
the pH at which you have 50% of the conjugate acid & 50% of the conjugate base
• when the pH of a solution equals a molecule’s pKa, that solution can best absorb changes in pH (addition of other basic or acidic molecules)
• [pKa is when the concentration of the acid form of a molecule = the concentration of the base form of the same molecule]
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At what pH will a buffered solution be most resistant to changes in the pH after the addition of acid or base?
when pH ~ pKa
• this is when the pH is most resistant to change upon addition of OH- or H+
• relatively large additions in the amount of base (or acid) produce only small changes in pH → maximal buffering capacity
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What form of a molecule predominates when the pH value is lower or higher than the pKa?
pH < pKa → acid form of the molecule predominates
pH > pKa → base predominates
[base is form of molecule with fewer hydrogens]
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What is a major buffer inside cells?
phosphate (pKa ~ 7)
H2PO4- ↔ H+ + HPO42-
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What is a major buffer in the blood?
Carbonic acid/Bicarbonate
H2CO3 ↔ H+ + HCO3-
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Proteins make a significant contribution to the buffering capacity in cells and plasma. What part of proteins allows them to act as buffers?
• titratable groups on the amino acid side chains
• lots of amino groups & carboxyl groups (the AAs cyteine & histidine are especially good buffers)
• pKa of these groups differ depending on the side chain
• proteins have a considerable buffering capacity because the protein concentration in cells & plasma is quite high
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In terms of breathing, what causes the pH of blood to rise or fall?
• taking deep breaths → ↑ pH (CO2 is released)
• pulmonary obstruction/holding one’s breath → ↓ pH (CO2 builds up)
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How Aspirin Gets Absorbed
- • upon entering the stomach, Aspirin will be uncharged
- - stomach pH = 1.5
- - pKa of Aspirin = 3.5 [it’s a weak acid]
- - at a pH < pKa → Aspirin will exist in it’s acid form (more protons)
- - because of the acidic environment, Aspirin will be protonated (-COOH) & therefore uncharged
• as an uncharged molecule it can passively cross membranes
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Same Q: Aspirin is a weak acid at low pH and has a pKa of 3.5. Will more aspirin be absorbed in the stomach (pH 1.5) or in the intestine (pH 6.5)?
• when pH < pKa, molecule exists in its acidic form (RCOOH)
• when pH > pKa, molecule exists in its basic form (RCOO-)
• non-ionized molecules cross cell membranes passively whereas ionized molecules do not, more aspirin will be absorbed in the stomach than in the intestine
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Isoelectric Point (pI)
the pH at which a compound is electrically neutral, aka the net charge is zero
to find, average the 2 pKa's surrounding the isoelectric species
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Proline
contains a ring that limits its flexibility & makes it incompatible with alpha helix formation (called the PRO helix breaker)
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Tyrosine & Tryptophan
• both absorb UV light (280 nm) and makes protein detection easy
• tryptophan has a non-polar R group
• tyrosine has a polar R group
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Cysteine
can form disulfide bonds between S's at the end of their R group
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Which amino acid is capable of forming disulfide bonds?
Cysteine: disulfide bonds are most often found in oxidizing environments such as outside of the cell
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Which amino acids are positively charged at neutral pH?
arginine, lysine, histidine
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Which amino acids are negatively charged at neutral pH?
aspartate & glutamate (ate = negatively charged)
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Which of the 20 standard amino acids are aromatic?
phenylalanine, tryptophan, and tyrosine (all have benzene rings)
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Regions of proteins that interact with DNA are usually __________ charged:
Positively charged, because DNA carries a negative charge
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