2 - primary structure + purification Flashcards
whats an oligopeptide
synthetic, series of aa joined by peptide bonds
whats a polypeptide
long chain of aa (natural)
whats a conjugated protein
protein + prosthetic group(something that isnt protein)
how long are most proteins
100-1000
what ultimately determines the structure and function of polypeptides
primary sequence
what is Mr
relative molecular mass/molecular weight, same as Da but no units
what is 1 Da
1/12 of Carbon 12
what is Da of free aa
128
what is Da of residue
110
does primary sequence information include the disulfide bonding pattern
Yes
but they are still i think 3 structure
which terminus does methylation occur
C
which terminus does amidation occur
C
which terminus does acetylation occur
N
which terminus does acylation occur
N
what does methylation do to charge
makes it no charge
what does amidation do to charge
makes it no charge
what does acetylation do to charge
makes it no charge
what does acylation do to charge
makes it no charge
what is diff with acylation and acetylation
acylation is general (COR group added) but acetylation adds COCH3 (R=CH3)
what does formylation do to charge
makes it no charge
do proteins with different functions have the same sequence
no
do proteins with same functions have the same sequence
yes or very similar
what happens when you change aa sequence (to structure and function)
changes them
what are 2 things that a sequence can predict
structural information and cell localization
what is the most useful way to analyze primary sequence
compare with other sequences do generate similarities in structure and/or function
what is homology modelling
you can build a model if a sequence if it is close to another amino acid sequence
what are invariant residues
amino acids which do not vary at all between a set of sequences
what is the significance of invariant residues
critical in protein function
what are conservative substitutions
when one amino acid is substituted with another similar one
what are non-conservative substitutions
when one amino acid is substituted for another with dissimilar characteristics
what is the significance of non-conservative substitutions
may reflect differences in functions between two proteins or less important/critical locations in structure
what does it take for an amino acid replacement to be considered conservative
if there are at least 2 similarities (polarity, size, functional groups…)
what is sequence alignments
2 or more aligned to make the best or closest match
-largest number of invariant or conservative substitutions
what is % identity
identical positions/total positions
what is % similarity
conservative positions/total positions
is % similarity or % identity bigger
% similarity
what does a -# in matrices for scoring alignments mean
low likelihood that you would find that aa swapped out in another sequence
what does a +# in matrices for scoring alignments mean
high likelihood to be swapped out for in another sequence
what is a “gap” in protein structure
2 sections of aa sequence that may align well but have different lenghts
what causes gaps in protein structure
insertions or deletions at the genetic level
what do gaps correlate to in the actual protein structure
loops on the surface, or the ends that change (C or N terminus)
what happens with a mutation in a loop vs middle of helix
loop- likely ok, keep function
helix-bad
what happens if the amino acid mutation is non-functional
it may be detrimental and not retained
what happens if the amino acid mutation is conservative
neutral so the change may be retained
are all aa positions equally sensitive to change
no
some more than others
what does the degree in sequence between polypeptide correspond to
length of time since divergence
what are homologs
two proteins which share a common ancestor
what are “any two proteins which arose out of a single gene”
homologs
what are 2 ways that homologs can arise
duplication or speciation events
what are orthologs
homologs (two proteins which share a common ancestor) which arise out of speciation events
what are paralogs
homologs (two proteins which share a common ancestor) which arise out of gene duplication events
how do orthologs compare in different organisms (function wise)
carry out the SAME function (speciation)
how do paralogs compare in different organisms (function wise)
may have the same or different functions (gene duplication)
are beta-globin and alpha-globin para or ortho logs
paralogs (gene duplication)
is human beta-globin vs mouse beta-globin para or ortho logs
orthologs (speciation)
do you use orthologs or paralogs to make evolutionary trees
orthologs
how can you purify proteins based on solubility
salting out
how can you purify proteins based on ionic charge
- ion exchange chromatography
- electrophoresis
- isoelectric focusing
how can you purify proteins based on polarity
hydrophobic interaction chromatography
how can you purify proteins based on size
- gel filtration/ size exclusion chromatography
- SDS PAGE
- dialysis
how can you purify proteins based on binding specificity
affinity chromatography
what do you want to minimize during purification
denaturation and degradation
what are the 3 most important + 2 other things to control in protein purification
pH (use buffers)
temperatures (slow cold)
enzymes (inhibit protease maybe)
then adsorption (how protein sticks to glass, plastics) long term stability (oxidation, denaturation)
how do you directly monitor concentrations of protein of interest vs. other compounds
enzyme activity! and spectrophotometry (if protein is associated with specific profile, like color)
how do you indirectly monitor concentrations of protein of interest vs. other compounds
antibody-linked/immunoassays (use if protein is antigen for specific antibody)
definition of activity
ability to convert substrate into produce, usually indicated in units
what does 1 unit=
1 μmol substrate converted per min
what is specific activity definition
activity as a fraction of total protein
what is specific activity formula
units/mg total protein
what happens to specific activity as time goes on in purification
RISE (less contaminants)
what happens to activity as time goes on in purification
LOW (less % recovery)
when is spectroscopy good
specific absorption in some proteins (prosthetic groups, hemoglobin, cytochrome)
when are immunoassays good
non-enzymatic proteins or ones with no appropriate enzyme assay
when can you use immunoassays
if there are appropriate antibodies available
how does centrifugation work / what it do
fractionation of compounds based on mass/sedimentation rate
what is differential centrifugation / how does it separate
separates compounds by sedimentation rate
what kind of compounds sediment fastest in differential centrifugation
large proteins sediment faster than smaller ones (experience less friction and SA or something)
what is density/isopycnic centrifugation
separates compounds by density
what kind of process is density/isopycnic centrifugation
equilibrium process - density gradient in sample tube determines final location/position
how does density/isopycnic centrifugation separate/work
density gradient in sample tube determines final location/position
does time matter is differential centrifugation
ya think so
you can spin too much i think
does time matter is density/isopycnic centrifugation
no, once it hits equilibrium then it will always stay there
what is solubility/ precipitation in protein purification
some proteins solubilize/precipitate under different salt concentrations and pH
what causes salting in/salting out
some proteins solubilize/precipitate under different salt concentrations and pH
what is a common salt used in solubility/ precipitation in protein purification
ammonium sulphate (NH4)2SO4
what is often combined with solubility/ precipitation in protein purification
centrifugation
what kind of technique is dialysis
size based
how do you use dialysis / why
to remove small molecules from preparations (anything smaller than the pores in the membrane will go through the membrane and leave the bag)
what does column chromatography allow for
differential separation of compounds based on physical characteristics
what does rate of travel through a column depend on
relative affinity for the solid/stationary or liquid/mobile phase
what are some types of stationary phase
gel, beads, resin, polymer
what happens to migration rate with affinity to the solid phase
slows migration rate
what happens to migration rate with affinity to the liquid phase
increases migration rate
what is the mobile/liquid phase
the fluid that constantly flows from one end of column to the other
when do you use cation exchange
when you have a positively charged protein that you want to purify
when do you use anion exchange
when you have a negatively charged protein that you want to purify
what is the setup in cation exchange
stationary phase is negative, more positive are attracted to beads (elute slowly)
what is the setup in anion exchange
stationary phase is positive, more negative are attracted to beads (elute slowly)
how do you increase more release from the ion exchange column
increase salt concentration to elute things that bind weak, the more salt to elute medium binding, etc
what does the charge in the ion-exchange chromatography depend on
protein pI and pH of buffer
what pH do you use to elute in cation exchanger
0.5-1.5pH below the pI of interest (want it to be positive)
what pH do you use to elute in anion exchanger
0.5-1.5pH above the pI of interest (want it to be negative)
why dont you want a big gap in pH and pI in cation or anion exchange
too big gap = denature (too charged)
what is another name for size-exclusion chromatography
gel filtration
what is another name for gel filtration chromatography
size-exclusion
what is the stationary phase in size-exclusion
porous beads with small openings
what moves fastest gel filtration chromatography (size exclusion)
mobile phase moves faster around the beads than through them
does size-exclusion chromatography/gel filtration denature
no, doesn’t wreck 4 structure
when do you use affinity chromatography
when you have a specific protein of interesting binding a specific ligand
what will bind to the stationary phase in affinity chromatography
only proteins that bind to a specific ligand (ligand is covalently bound to stationary phase)
what happens once you add the ligand in solution in affinity chromatography
it will allows the protein to elute from the column (bound to free ligand instead of column)
how can you engineer a protein to help it stick in affinity chromatography
things like His tags or GST fusions
are engineered proteins for affinity chromatography supposed to change original structure and function
ideally not
what are his tags
6-14 His inserted into polypeptides usually a N or C terminus
why do you use His tags
to increase protein solubility or help to bind to columns (nickel)
what kind of column is good for his tagged proteins
nickel
how does His bind to Nickel
coordination bond from imidazole ring to metal ion
how can you elute things from column with his tags
changing pH or adding free imidazole
weakens protein/column activity
how do you wanna change pH when using his tags and nickel
lower pH so it will be protonated and cant coordinate
why do you add free imidazole when using his tags and nickel
outcompetes with his in protein so it leaves the column
desalting columns remove salt and other small molecules from protein solutions. what kind of chromatography is it
size exclusion bc small
what does electrophoresis do
separates proteins based on charge/mass ratio and size
what do you do to proteins before electrophoresis
add SDS DTT and then heat
what is and does SDS do
anionic detergent which denatures proteins and makes a consistent charge to mass ratio
what is and does DTT do
reducing agent to disulphides, allows for more complete denaturation
how long do you hear proteins before electrophoresis and how hot
why
95 C for 5 mins
for complete denaturation
how does SDS bind to aa and how often
1SDS binds for every 2 residues
why is SDS good to measure molecular weight of proteins
since it gives them all a consistent charge to mass ratio and denatures, it separates them based on mass (small migrate quickest)
what is added after electrophoresis
coomassie blue
where does coomassie blue bind
to proteins in the gel so that each polypeptide can be seen as a descrete band
do what point are proteins denatured in SDS PAGE (primary secondary tri tert which one)
primary and some secondary
how do you graph the molecular weight estimation via SDS PAGE (what are axes)
Log Mr vs migration distance - it will be linear
using SDS PAGE, calculated Mr (based on migration) is 10% lower than actual Mr why
smaller # means it moves faster
so the protein may be more negative than expected
what is western blotting
visualization of proteins based on interactions with antibodies
is western blotting sensitive or specific
very
when is western blotting performed and why
after SDS PAGE to locate proteins of interest amongst other present proteins
what is the western block TRANSFER process
run current perpendicular with - cathode above gel and positive anode undergel and membrane under gel (top to bottom:-cathode , filter paper, gel, membrane, filter paper, +anode)
what attaches to the protein in western blot
a primary antibody to specific antigen on protein
what attaches to the primary antibody in western blot
secondary antibody with a detectible portion (like enzyme to catalyze a visible reaction-color or luminescence)
what is added to the surface of the membrane in western blotting
blocking agent to block unoccupied sites on membrane (so like skim milk powder)
what is the intensity of the western blot band used for
quantifying concentration/amount of protein present
how does isoelectric focusing work
gel has pH gradient, samples migrate in a direction determined by pI/charge
what end is the positive and negative cathod anode put in isoelectric focusing
positive cathode at top because anything more negative will run up (low pH on top)
is low pH on top or bottom in isoelectric focusing
top
is isoelectric focusing based on time
no, its impossible to run for too long (equilibrium based technique)
what are 2 equilibrium based technique seen in protein purification
isoelectric focusing
density/isopycnic centrifugation
what happens in 2D electrophoresis (axis)
isoelectrical focusing (pI) vs SDS PAGE (molecular weight)
what should happen to the amount of bands as you purify
there should be less and less
