Download MCB Lecture 2 – Amino Acids and Proteins

Lecture 2 – Amino Acids and Proteins
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Primary Structure – linear sequence of amino acids, from –NH2 terminal to –
COOH terminal
Secondary Structure – 3-dimensional structure caused by H-bonding
between peptide bonds.
o Alpha Helix – Hydrogen bonding happens 4 amino acids apart. No
proline (destabilizes) or glycine (too flexibile).
o Beta Sheet – Can be anti-parallel or parallel. (no clear difference in
stability)
o Beta Turn – 180 degree turn involving 4 amino acids in turn, and one
H-bond per every 2 AA. Has a lot of Proline and Glycine.
Tertiary Structure – 3-dimensional structure based on non-covalent
interactions (Van der Waals, H-Bonding, Electrostatic Interactions) and
covalent bonds (Cys-Cys bond – disulfide bond)
o Protein Domain – a tertiary structure where different sections of one
polypeptide chain can take multiple secondary arrangements.
Changing domains has been the cause of evolutionary differences in
species. Example: actin fold where ATP binds.
Quaternary Structure – Multiple protein subunits bound together to form a 3dimensional shape.
Hydropathy Plot – A plot that determines how hydrophobic particular amino
acids are in a chain. Above the x-axis is most hydrophobic, and below the xaxis is hydrophilic.
What is the maximal UV absorption of a protein? 280
What is the maximal UV absorption of DNA? 260
To form a peptide bond what happens? Hydrolysis – water is removed.
What percentage of “likeness” do two polypeptide chains have to have to be
considered structurally similar? 25%
Orthologs – Same function, different species
Paralogs – Same species, related sequence, different function.
Size-Exclusion Chromatography – Gelfiltration where beads trap smaller
proteins and larger ones elude out first.
Ion-Exchange Chromatography – Beads in the column are charged (-charged)
so the same charge eludes first (-charge) and opposite charge is attracted and
trapped (+charge)
Affinity Chromatography – Beads have ligand attached that is specific to the
desired protein. The desired protein stays in the column while unwanted
proteins elude. Adding another molecule with a higher affinity to the ligand
will release desired protein from column.
Gel Electrophoresis – Gel column that separates proteins based on size
(smallest travels fastest). Negative charge at top, positive charge at the
bottom so negatively charged DNA runs towards + charge. Agarose for DNA
and Polyacrylamide from protein.
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o What reagents denature protein for Gel Electrophoresis? SDS and
Mercaptoethanol.
o How can you estimate the molecule weight of a protein? Add a
protein with a known weight to another well of the column and
compare migration.
Isoelectric Point – The pH where the molecule you are testing is neutral.
Isoelectric Focusing – Establishing a pH gradient in a gel. Adding a TON of
different proteins. Once they hit neutral pH, they stop traveling. Compare
according to their isoelectric points. Add gel to an electrophoresis to then
separate based on size and identify.
How do determine the affinity one protein will have to another (like an
enzyme)? The number of non-covalent bonds determines the affinity.
If Kd > Ka  Dissociate
If Ka > Kd  Associate
If Kd > Keq  Dissociate
If Kd < Keq  Associate