Midterm 2 Flashcards
(113 cards)
are naturally occurring amino acids D or L isomers
L
what are the angles between C alpha bonds
its sp3 so 109.5
At what ph is zwitterionic form present
~ 7
Assuming the side chain has no ionizable group, at approximately what pH will the majority of this free amino acid have a net positive charge?
Most of the amino acids will be positively charged below pH ~2 (-N+H3; -COOH) and will be negatively charged above pH ~9 (-NH2; -COO-)
what amino acid forms a covalent disulphide bond
Cys
what amino acids are considered to be helix breakers
Pro
Gly
Name two features that all amino acids except glycine have in common?
chiral
a methylene group (CH2) attached to alpha C
At approx. what pH is the majority of a free amino acid with no ionizable side chains a zwitterion?
pH 4-7
At approx. what pH will most of
a free amino acid with no ionizable side chains have a net negative charge?
> pH 11
At approx. what pH will a free amino acid with no ionizable side chains be non-ionized?
No pH
at pH <2, the it will have a net positive charge (alpha- amino and alpha-carboxyl groups both protonated)
and at pH >8, it will have a net negative charge (alpha- amino and alpha-carboxyl groups both deprotonated).
At what pH are the concentrations of histidine (+) and histidine (zwitterion) equal?
[HA+] = [A] when pH = pKa
Why is histidine considered both an acid and a base?
At neutral pH (7) there will be both HA+ and A present (although there will be 10X more A than
HA+); HA+ can act like an acid and lose a proton and A can act like a base and gain a proton.
where is a possible place a covalent bond could occur between two side chains
cys
Why are there rarely peaks in the lower right quadrant of a Ramachandran plot?
The angles in the lower right of the plot would lead to steric clashes between side chains and are not stable/favored so are not usually seen in proteins
How does the H-bonding pattern differ between alpha-helices and beta-sheets?
In alpha-helices the H-bonds occur between residues within the same alpha-helix, from one carbonyl oxygen to an amide nitrogen 4 residues away,
whereas in beta-sheets the H-bonds are between beta- strands.
In antiparallel beta-sheets, each amino acid donates an H-bond and accepts an H-bond from the same amino acid in the neighbouring beta-strand, whereas in parallel beta -sheets, the amide nitrogen and carbonyl oxygen of one amino acid H-bond with 2 adjacent amino acids on neighbouring beta-strands.
Why must the side chains on the outside of a beta-barrel membrane pore be non-polar?
Because they are in contact with the hydrophobic lipid bilayer.
Compare and contrast turns and loops?
Turns are short - 3-4 amino acids – and stabilized by a H-bond; loops are longer than 4 aa and irregular in structure (i.e. they vary from one loop to another, amino acids do not have regular or predictable phi/psi angles, or a regular pattern of H-bonds)
But both are typically present on the protein surface, both connect repeating secondary structures (alpha-helices, beta-strands) and both typically reverse the direction of the polypeptide chain.
Briefly explain the Levinthal paradox and how it is resolved. What determines the final native conformation of a protein?
There are such a vast number of different conformations a protein can adopt that it cannot possibly sample each conformation in search of its native, most stable structure in less than a second (the time it takes for a protein to fold). Instead, proteins are thought to follow a folding path or a “landscape” leading to a thermodynamically stable fold (this landscape may involve transiently stable intermediates). The hydrophobic effect is a strong driver of protein folding. The amino acid sequence of the protein dictates its final structure.
What is the function of a chaperone?
A chaperone assists protein in folding by shielding its hydrophobic regions from improper associations that may lead to aggregation. (Some chaperones also help transport proteins from one part of the cell to another, keeping them unfolded until they reach a site where they can fold; some help proteins assemble into quaternary structures, help aggregated proteins to fold, prevent damaged proteins from refolding …)
Briefly explain the principle behind each type of column chromatography and explain how the protein of interest is eventually eluted.
- ion exchange chromatography: proteins with a net charge bind to a gel matrix of the opposite charge; they are eluted with high [salt], the ions of which compete with the electrostatic interactions of the protein and the matrix
-size exclusion chromatography: porous gel beads trap small proteins while larger proteins pass between the beads; this slows small proteins down; eventually they will elute from the column as more solution is passed through it
-affinity chromatography: a ligand known to bind the protein of interest is attached to the gel matrix; the protein will bind to this ligand and other proteins will pass through the column; the bound protein can be eluted with free/soluble ligand
Explain what causes proteins to migrate from the well to the bottom of the gel in SDS-PAGE. Which proteins get to the bottom first? Why?
Proteins are denatured with SDS, which coats them with a negative charge. When they are loaded into a well of a gel and an electric field is placed across the gel the negatively charged proteins will migrate toward the positive terminus. (Note, this is sometimes called the cathode, sometimes the anode – just know that it is the positive pole to which the negatively charged proteins migrate.) The smallest proteins get to the bottom first because they are not retarded by the acrylamide cross-linker in the gel. (Note that this is the opposite of what happens in size exclusion chromatography, where small proteins are retarded because they get caught in the pores of the gel beads, and large proteins pass right between the beads and elute first.)
Compare and contrast myoglobin and hemoglobin with respect to structure, O2 binding, regulation.
Both small proteins that are mostly -helical, 8 helices per subunit, have a hydrophobic cavity that binds heme and oxygen . Myoglobin: monomeric, 1 heme, in muscle cells, binds 1 O2 molecule tightly, binding not influenced by pH, CO2 concentration or BPG. Hemoglobin: tetrameric, 1 heme/monomer, in RBCs, binds 4 O2 molecules weakly, cooperatively, O2 binding is influenced by pH, CO2 concentration, BPG
There are 2 histidines involved in coordinating the heme iron for both myoglobin and hemoglobin. How do their roles differ?
His F8 directly coordinates with the heme Fe2+, whereas His E7 stabilizes O2 when it is bound.
Briefly explain how oxygen binding to the heme of one hemoglobin subunit increases the affinity for O2 at the other subunits (ie. how the subunits work cooperatively). Be precise about where the oxygen binds, what the effect is …
Oxygen binding to the heme iron brings the iron into the plane of the heme protoporphyrin ring. This pulls along His F8 (8th residue on -helix F), which pulls helix F, whose C-terminus contacts a neighboring subunit. The structural change at F induces a conformational change in the neighboring subunit , causing the subunits to shift and rotate relative to each other . This change in the hemoglobin tetramer represents the transition from the “tense” or “T” state, which has a low affinity for oxygen , to the “relaxed” or “R” state, which has a higher affinity for oxygen . Thus, binding at one subunit enhances the affinity of the other subunits for oxygen.
