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Apolar residues
Is non polar.
Glycine- H
Alanine- methyl group
Valine- isopropyl
Leucine- isobutyl (iso is where butyl, etc is attached)
Isoleucine- sec-butyl
Methionine s-methylethyl
Proline- pyrrolidine
Phenylalanine- benzyl (phenyl)
Trypophan- indolylmethyl (indole)
2.
-
Polar residues
Molecules that have a polarized bond.
Charge separation- dipole in their R group.
Serine- hydroxymethyl
Threonine- hydroxyethyl
Asparagine- amidomethyl
Glutamine- amidoethyl
Tyrosine- hydroxybenzyl
Cysteine- sulfhydrylmethyl
Understand to be given a value and see where the reaction will go.
Above 10.5 will ionize bc at ph 10.5 will be 50/50 percent. 50% oh and
50% o- . above ph 10.5, more will be o-. will have more protons.
at ph 10 (go down) then will have more protons, will push rxn backwardswill push o- to neutral form.
3. Charged residues
- Lysine
- Arginine- has guanidine group attached. Only aa with guanidine. Bridge
is propyl.
- Histidine- has imidazole functional group. Is attached to the amino acid
backbone.
- Aspartic acid- carboxyl r group
- Glutamic acid. carboxyl r group.
- Given pka is 6 or 8. Which way rxn goes.
- Basic residues- accept a proton. At 10.5 50% protonated 50% neutral.
Ph 7.4/7.5 is predominantly one state- is protonated because is going
down. Now have enough protons will push it to the right.
- Acidic acid go to acetate plus a proton. Ph4- is 50/50, at ph 2 will be
protonated all acid. At ph7 will be predominately in the unionic form.
- Histidine- only basic or neutral- never acidic. Ph 6- 50/50 percent
protonated and neutral. Phh 7.4- will be neutral, below ph 6 is
protonated.
- Ph<pka- r group is protonated.
Dipolar amino acids- Zwitterions
- Pka of amino and carboxylic groups are 9 and 2.
Nomenclature- Alpha amino, alpha carboxyl.
- Greek alphabet extends.
- Ex. Epsilon amino group was replaced.
- Two bonds- peptide and disulfide bonds.
Peptide linkage- condensation
- When you conjugate things the nomenclature changes.
b. Stereochemistry
- All amino acids are L
- Most sugars are D
Polarized light
- Is perpendicular and is in phase.
- Light has 2 electromagnetic wave. B is always perpendicular
- E can oscillate in all direction- is nonpolarized.
- E can be made to oscillate vertically or horizontally or elliptically.
- Polarization of light refers to the direction of oscillation of E.
c. Derivatives
- Altering PTM can cause an entire protein to change. Can make it active
inactive, etc.
- PTMs- phosphorylation, methylation, acetylation.
- Are reversible.
- Are molecular switches to alter and modulate protein function.
- Free aa can also be modified.
- 1. Serine- phosphorylated derivative
- 2. Glutamate- carboxylated derivative.
- 3. Proline- hydroxylated
- 4. Histidine- methylated. After methylated the histidine is neutralized,
side chain cant be protonated anymore.
- 5. Lysine- is acetylated. Acetylation neutralizes or reduces the charge.
-
Neurotransmitters
Decarboxylated glutamate
Histamine- carboxylate missing
Dopamine
Tyrosine
2.2b
-
Protein purification
work with proteins in isolation
want to purify it and isolate it so no other proteins are with it
need to purify proteins in order to understand their biophysical and
biochem properties- thermos, kinetics, catalysis, structure.
- Want to know the structure of proteins- if we knew what the protein
looked like then can infer its function- not true.
- Look at protein unique structure and chemistry in order to separate it
from other molecules
- Ph temp and pressure is important. These affect protein function and
stability
- Isoelectric point- pI is simply the pH at which a given protein carries
no net charge. Need to know this or else protein will precipitate out. If
no net charge then protein will precipitate out and not solubilize.
Isoelectric point
- Dont need to know pIs
- Ex. Myoglobin or hemoglobin wont have charge at neutral pH 7. Equal
number means they have a equal number of positive and negative
residues.
- Net charge is sensitive to pH.
- Proton concentration changes- the equilibrium shifts. Change pH =
change its state.
- pI is the pKi (only refers to the charged aa- ionizable ones)
- N= number of charged residues.
- Add up pKis and divide by N.
- Protein purification should be done away from the pI or else it will
precipitate protein- enzymes will become inactive. Have to know pI of
protein from its aa sequence.
- If purifying myoglobin or hemoglobin want to work away from 7. Want
to work ideally above 8 or below 6.
Salting out
- Outdated method
- Precipitates proteins
- Generally dont want to do this.
- Salt ions compete with protein molecules for bulk solvent.
- Add salt and make protein come out.
- Salt interact with water and will compete with the protein for the water.
- Crash- coming out of solution.
- Protein is colorless liquid- that means protein is soluble. Proteins can
be membrane or water soluble. All about water soluble. This section.
- Water sol- proteins in the cell, hemoglobin, myoglobin etc. not
membrane proteins like gpcrs.
- Increase salt concentration- salt competes with protein for water- will
therefore have a high concentration- not enough solute available to
compete. Salt will come in and push protein out.
- Want to be right at the spot of pI- is where the protein is least soluble.
Proteins least soluble at their pI.
- Most common used salt is ammonium sulfate- is KOSMOTROPHIC.
(dont need to know) has a high affinity for water and can take away a
lot of water.
- Makes a hydration shell- bigger hydration shell means it can take away
more water.
- Protein in solution- yellow protein is the one we want.
- Different proteins have different solubilities.
- Green more soluble than purple.
- Solution has no salt. Will spin out, least soluble protein will sediment to
the bottom by centrifugal force.
- Increase the salt concentration, remove purple, next least soluble
protein will sediment. Will come out as precipitate. Then only yellow
protein remains.
- Exploiting out solubility
Ion exchange chromatography IEC
- Are exploiting the charge, proteins can be negative, neutral and
positive depending on pH.
- Is ion exchange
- Ion exchange column. Column is attached to exchangers. If protein is
negative- charge protein with DEAE. Protein is covalently bonded to
this. Anion exchanger.
- Protein is positive use CM- cation exchanger, will bind cations.
- Our protein is negative, have column with DEAE- anion exchanger.
Have solution with a bunch of proteins
- Apply to column, will run through. Proteins that are neutral or positive
will run through- charge isnt compatible so will not stick. Your protein
has neg charge so has high affinity and will bind and have electrostatic
interactions.
Size
-
Add salt (nacl) cl ions will bind to thing, na ions will bind to the proteintarget protein can now come off the column.
Exploiting net charge of protein and column is charged, your proteins
will be selective.
exclusion chromatography
Exploiting out size of protein as well as its conformation (shape)
Proteins can be round, globular
Apply protein to a column- use a gel with small tiny pores.
Is a filter- molecular sieve. Has a proe matrix with tiny pores. Protein
mixture has different sizes. Small proteins enter the pore, large
proteins dont enter.
Smaller they are- more likely to enter.
As it goes through- small molecules will enter the gell. Larger ones will
bypass.
Small- enter gel matrix, will slow down.
Larger molecules will run through faster because they werent impeded
Small- motion down the column isnt impeded.
Two proteins can be same number of residues- globular protein- may
enter column, rod may not enter column
Smaller impeded
Larger come out faster.
Affinity chromatography
- Have a ligand that has an affinity for your protein of interest.
- Ligands can bind to proteins- can engineer proteins.
- Clone the protein= express in bacteria outside of body.
- Clone the protein with a Gst tag onto the protein= are called fusion
proteins- your protein plus a tag. Marker can be a gst tag or a histidine
tag (6 residues)
- Gst- is a protein- if protein is not soluble will enhance the solubility of
the protein- gst is very soluble and stable and will keep the protein in
the solution. However protein is tagged so can affect its behavior.
- If you do gst tag- charge the column with glu.
- Immobilize ligand onto column
- Bacterioize column = apply impure protein onto column- protein will
bind to the gst. All other proteins will just go thru.
- If have histidine- charge column with nickle- his tag can coordinate
with nickle ions. Strong affinity rxn. All will go thru. After go thru make
sure protein come off column
- Gluthatione if use gst to get protein off column
- Imidazole- allows ur protein to come off column if use his.
Quality control
- Purity and yield
- Purity- visually analyze- use SDS page- gives you an idea if there are
any contaminants
- Sds denatures protein- use tiny bit of purified protein- denature it.
- Look at protein concentration
Sds concentration
- Electrophoresis- charged molecules migrate in a fluid medium like gel
under the influence of electric field.
- PAGE- trying to separate proteins on the basis of their mass.
- Charge and shape arent depended on molar mass of protein. Get no
resolution- two molecules can migrate the same if they are similar.
- Separating molecules on basis of size with PAGE.
- Electrophoresis- highly charged.
- Coat protein with sds- will denature them and make them all rods. All
proteins small and large will have identical q/m ratios. Since they can
bind more SDS- no matter size of protein. Cant separate proteins bc
have same q/m ratio in sds page.
Gel contribution
- All the proteins get same force and same weight.
- If bigger- get more drag- is impeded- more slower. Gel separates them.
- Separate out bc the bigger proteins cannot move. So big bc the gel
impedes their movement. Tiny proteins will run thru the gel.
Beer lambert law- measure protein concentration. Can calculate or have
epsilon value
a- Measure.
Can calculate concentration.
Units.
2.2c Protein sequencing
- First protein in 1955
- Make larger protein to smaller fragments- make them overlapping
fragments
- Step 3-5 for all proteins
1. Identify n terminal residues
a. Only for multisubunit protein
b. React polypeptide chain with dansyl cl
c. Breaks residue
d. N terminal is a derivative
e. Purifcy with hpl- is hydrophobic. Different molecules have
different rxns.
2. Separate subunits
a. Ex. Insulin , disulfide bonds- most common lab agent is
reducing agent.
Hb is a oxygen
Mb serves as a reservoir- stores oxy.
M has a hyperbolic o binding curve.
H binds in a sigmoidal o2 binding curve and serves its function
Ph and bpg- these two affect the ability of h to bind oxygen.
Allosteric= change the conformation of hemoglobin.
Hemoglobin is a tetramer
Myoglobin is predominately in the muscle
Plays a role to store o2 and to facilitate the release of hemoglobin
Will snap o2 off hemoglobin and will help it relay to tissues
Function of both is related to a cofactor- heme.
Prosthetic group- a specific type of cofactor.
- share very little identity or similarity in amino acid sequence. Structures are
really similar
- proteins that have little in common can have same form
- structures look like alpha form with different helices- a through h.
- stretch them out- helices are a to 8.
- Bring back together and fold it- tertiary structure
- Ab is a customary can be called alpha or beta helix.
- Myoglobin is a single polypeptide- is monomeric
- Have 4 of them- 2 alpha and two beta- is important to hemoglobins
function.
- Macrocyclic ring- dont have extra moieties- have 4 pyrol groups.
- Proferin center of heme lies iron.
- Histidine from helix f.
- Helix is a-h in hemoglobin.
- Proximal face- side pointing away from where the oxygen binds.
Myoglobin
- In addition to oxygen , other gases can compete with oxygen and can
bind to myoglobin. Can deprive o2 from binding.
- Allosteric- when something binds to a protein- binding on one side
causes a conformational change on the other side.
- Allosteric- proteins when u look at their structures- are very flexible.
Ligand binding causes a conformational change.
- Is advantageous to proteins because dont have a pocket for binding
ligand.
- Dont have final shape fully formed until they see the ligand in order to
fit.
- Can go conformational change rather than before binding.
- Allostery key role in binding of o2 to myoglobin.
- K is concentration of two reactants divided by the products.
- Myoglobin bound to oxygen- is equal to the product.
- Concentration divided by k.
- At equilibrium- concentration of free myoglobin is equal to bound- is
concentration of the ligand where protein is concentrated.
- Less we need, the stronger.
- In equilibrium- the lower the value the better because need less
concentration.
- Y is the amount oxygen bound to the myoglobin.
- Myoglobin can only be free or bound.
- Oxygen is a gas- express in partial pressure.
- At higher concentrations the curve flattens out.
- Curve has typical hyperbolic response- no oxygen
Hyperbolic curve.
- Curve flattens at around 10 torr.
2.5b
Competitive inhibition
Inhibitor resembles the substrate. Action by binding to the substrate.
Competes (i) with s to bind to e for active site
Cant be converted to p
Ki = reactants/ products.
Will reduce the conc of available enzyme for substrate binding= Km is
increased. By alpha
Increased because inhibitor is competing with the substrate (thinks
that there is less enzymes- need much more substrate in order to
compete with inhibitor)
Alpha is greater than or equal to one.
Increased so multiply the term.
Once reach vmax- need a lot of s.
Lineweaver
o For competitive inhibition- increase by alpha. Multiply by alpha.
o Intercept is a constant. Incr inhibitor conc- have lines at 1/vmax
cross there.
Uncompetitive
o Inhibitor doesnt compete with substrate
o Substrate is noncompetitive
o Only binds to the ES, not the free enzyme!