Midterm 2 Flashcards
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.
Fetal hemoglobin has 2 gamma subunits in place of the beta subunits found in maternal hemoglobin. The gamma subunits have a serine in place of His143. Briefly explain how this allows fetal hemoglobin to outcompete maternal hemoglobin for oxygen binding.
Fetal hemoglobin binds to BPG with a lower affinity than maternal hemoglobin so it is more likely to bind to O2 - out-competes material hemoglobin for O2 binding. (2) (Maternal hemoglobin binds to negatively-charged BPG via 3 positively charged amino acids on each of its -subunits, including His143 [plus the N-terminal amine group]. Fetal hemoglobin has a total of 2 fewer positive charges [1 less His for each subunit]. BPG binding stabilizes the T state, prevents O2 from rebinding.)
Why doesn’t BPG bind to myoglobin?
Myoglobin is monomeric – doesn’t have a central cavity lined with positively-charged residues to bind the negatively-charged BPG. (Also, BPG is in RBCs, not other cells.)
Provide an example of weak chemical interactions provided by chymotrypsin that stabilize (i) the substrate and (ii) its transition state.
(i) The substrate binding pocket provides hydrophobic and van der Waals interactions for the R1 aromatic/bulky hydrophobic side chain.
(ii) The amide nitrogens of Gly193 and Ser195 form the oxyanion hole that stabilizes the negatively-charged O in the tetrahedral transition states (one that forms after nucleophilic attack of the carbonyl carbon by the Ser195 alkoxide ion and one that forms after nucleophilic attack ofthe same carbon by water).
Briefly compare trypsin and subtilisin? (i.e. how are they similar, how are they different?
They are both serine proteases, using a catalytic triad (Ser-His-Asp) to hydrolyze peptide bonds, but they are not related in amino acid sequence or structure. They are distinct proteins that have converged to the same mechanism and function.
what is a primary structure
amino acid sequence
what is a tertiary structure
folded peptide chain
what is a quaternary structure
assembled subunits
what stabilizes folded protein structure
a variety of weak chemical interactions
and in some cases covalent (disulphide bonds) between cysteine residues
what holds amino acids together
peptide bonds
what is the structure of an amino acid
r group
carboxyl group
amino group
H group
what is the only
what are zwitterions
have a net charge of 0 at ~ neutral ph
have separate positive and negative group
what does the ionization state of amino acids depend on
the ph
when is zwitterionic form dominant
~ physiological ph (7.4)
why does gly improve flexibility of in peptide backbone
small side chain so litter steric hinderance
what is aliphatic mean
non-aromatic
hydrophobic , non polar
what happens when bulkiness increases in aliphatic amino acids
hydrophobicity increases
what is most hydrophobic molecule
phe
what moderates the hydrophobicity in tyr and trp
presence of electronegative atoms (N,O)
when aromatic rings stack, how do they interact
van der waals
where are aromatic ring stacks often found
inside
how does phosphorylation change properties of side chains
reversible
activates/deactivates proteins
signal transduction
what is a helix breaker do
disrupts repeating structure of alpha helix
why is proline a turn maker and what are the effects
side chain cyclizes to form a bond with backbone N
which provides rigidity
induces kinks - found at turns
what does cysteine do
form disulphide bonds
how does fact that cytoplasm is a reducing agent affect disulphide bonds
they cant form in reducing environment
when do disulphide bonds form
after folding to stabilize it
what interactions occur with hydrophobic (inside) proteins
van der waal
what sort of bonds to hydrophilic (outside) side chains take part in
ionic and h-bonding each other and the backbone
what does protonation (ionization state) depend on
pka and ph
what happens if pH<pka
= acidic = molecule will be protonated
what happens when pH>pka
basic = molecule will be deprotonated
what direction are amino acids written in
N to C
what do phi and psi represent
N - phi (circle)
C - psi (trident)
what is an alpha helix
repeated coiled structure stabilized by H-bonds
in alpha helices what is the distance between carbonyl o and N of amide that are H-bonded
i+4
what are the angles for psi and phi in alpha
phi: -100. psi : -60
why are gly and pro less common in alpha helices
helix breakers
what amino acids induce curvature
gly and pro
what direction to R groups point in alpha
outwards
what is an amphipathic helices
have hydrophobic and hydrophillic properties
alpha helix as a cylinder
where in Dna does alpha helices and beta sheets fit in
a - major grooves
B- minor grooves
how r groups of backbone arranged in beta sheets
trans
how are beta sheets stabilized
h bonds
what holds strands in beta sheet together
h- bonding
what are the angles for phi and psi in beta sheets
phi: -120
psi: 120
what is the beta - barrel structure of porin
forms a selective channel
outside hydrophobic
inside: hydrophilic
what is a beta sandwich
2 beta sheets are stacked
flattened beta barrel
what are turns
3-4 amino acids
charctertistic h bonding and phi/psi angles
what are loops
are variable in length (longer than 4 ) and irregular
what are beta turns (reverse turns)
form a tight turn in peptide backbone that reverse direction of chain
what stabilizes beta turns
h-bonds
what is a gamma turn
turn with proline at position 2 (makes sharp turn)
what does in vitro mean
in test tube
what does in vivo mean
not in cell
what can protein fold be stabilized by
disulfide bonds
what is native form
most stable state of folded protein
lowest energy
- delta g
what is a denatured protein
not in notice state
what are the three proposed models of protein folding pathways
hydrophobic collapse model
frame work model
nucleation model
what is the hydrophobic collapse model
protein collapses around its hyrdrophobic side chains and then rearranges
what is the framework model
folding starts with formation of elements secondary structures independent of tertiary states
these then assemble into tertiary state
what is the nucleation model
early formation of a protein folding nucleus that catalyzes further folding
why is protein folding positive delta S
hydrophobic effect
what do chaperones do
prevent misfolding
some proteins need them to reach native state
how does GroEL fold proteins
- GroES and ATP bind to GroEL trapping unfolded protein in folding chamber
- and it exits to lower chamber
- ATP hydrolysis causes conformational change to reset chambers and fold proteins
what are the 2 classifications of folded proteins
globular : complex roughly spherical shape
fibrous: elongated
what are domains
compact and locally folded regions
what does SDS -PAGE do
separates proteins by mass
Its an ionic detergent which denatures and binds to proteins to make them uniformly negatively charged
so when current is applied to system, proteins migrate towards positivity charged terminal
for analysis NOT for purification
what do DTT and beta - ME do
reducing agents, can be in buffer solution to reduce disulphide bonds
explain 2D get electrophoresis
1st dimension: separates proteins according to natural charge (no SDS)
2nd: separated by size (SDS)
explain X-ray crysrallography
expose protein Chrystal to xray (xrays are scattered by atoms in crystal)
scattered waves are collected and so the structure can be determined
what is immunoflouresence
antibody based technique to identify proteins
allows you to see them
is myoglobin affected by pH, [CO2] or [BPG]
no
is hemoglobin affected by pH, [CO2] or [BPG]
yes
how does hemoglobin bind to O2
tightly with prosthetic/cofactor heme which contains Fe2+
what kind of bond does hemoglobin form with O2
unstable, reversible
explain cooperative binding of o2
as hemoglobin binds to successive oxygen, affinity of subunit increases
this means hemoglobin is very saturated at high pO2 (lungs) and releases O2 when po2 is low (tissues)
what are the two States hemoglobin exists in
tense - deoxy (low o2 affinity)
relaxed - oxygenated (high o2 affinity)
how does BPG help stabilize hemoglobin T state
only binds to deoxy hemoglobin
prevents rebinding of released O2
helps release of O2 into tissues
what is Bohr effect
Co2 lowers the pH of blood, which causes hemoglobin to release O2
shifts dissociation curve to the left
cells with increased metabolism release greater amounts of co2
hemoglobin differences in fetuses
has low affinity for BPG
have 2 gamma sunsuits instead of the 2 beta subunits in normal
this makes hemoglobin positively charged = lower BPG affinity
allows fetus to compete for oxygen
what are the 2 models to explain enzyme active site
lock and key - perfect fit between substrate and enzyme
induced fit - (current model) - complementary but not perfect fit , then conformational change
what is an allosteric enzyme
has more than one active site
what are the two types of reversible enzyme inhibitors
competitive; resembles substrate and competes for same active site
non-competitive : inhibitor binds to a different active site, allowing substate to still bind, but cant reach transition state
what does an irreversible enzyme inhibitor do
binds tightly (either covalently or non-covalently) and inactivate them
what are substrate analogs
bind to enzyme but cant be “turned over” (turned to products)
what are transition state analogs
inhibitor that resemble transit state and cant be turned over
often used to crystallize enzymes
what are serine proteases
enzymes that hydrolyze peptide bonds using activated serine
proteases cleave (hydrolyze) peptide bonds
what amino acids do things in crymtrypsin
ser: provides nuc
his: acts as a base catalyst to activate Ser
Asp: stabilizes protonated his
