Flashcards in Ch 3 Deck (53):
what is the proteome
the full complement of proteins expressed from an organism's genome
what does the proteome vary with?
cell type, developmental conditions, environmental conditions
how does the size of the proteome differ compared to the genome?
what is the general strategy for protein purification?
Tissue --> disrupt cells --> crude fractionation --> fractionation
how do we recognize the protein we are looking for?
perform an assay to identify a unique identifying property of enzye.
what is specific activity
ratio of enzyme activity to the amount of protein in a the mixture
how does dialysis separate proteins
it separates on the basis of size using a semipermeable membrane with a specific molecular pore size. large molecules cannot pass and get eluted first
how does gel filtration work?
also separates by size: column consists of porous beads. large molecules flow faster and elute first
what is the advantages of salting out a protein
maintains protein in a negatively folded state, simple process, and fast process
ion exchange chromatography
separation is based on net charge at pH 7. if a protein has a positive charge, it will bind to the charged carboxylate groups: negatively charged proteins will not bind. protein is eluted by increasing the concentration of salt added to running buffer. positively charged ions compete with protein for binding
use a chemical group protein of interest has a high affinity for. Concanavalin A binds to glucose. add it to a column with glucose in it, then elute it with concentrated glucose
what is a his tag
a string of 6-12 histidine residues added to N or C terminus of a protein. binds nickel (II) ions.
how is a his tag used to purify protein?
protein with his tag is put through column containing Nickel II ions. protein is eluted with imidazole solution that binds to nickel II ions
movement of charged solutes through a gel in response to an electric field
chemically inert; polymerized acrylamide matrix of controlled pore size; allows separation of proteins based on mass and charge
how does size affect migration in polyacrylamide gels?
larger proteins migrate slower
smaller proteins migrate faster
what does Sodium Dodecyl Sulfate do?
negatively charged detergent used to denature proteins: 1 SDS/2 amino acids. results in a denatured protein with negative charge proportional to protein mass
what does mercaptoethanol and dithiothreitol do?
reducing agents that break disulfide bonds and help denature tertiary structure of proteins
electrophoretic mobility relationship to mass?
electrophoretic mobility of many proteins is inversely proportional to the logarithm of mass
what is isoelectric focusing?
separating proteins by electrophoresis based on native charge
what is native charge dependent on?
dependent on the number of acidic and basic residues
how is the gel made for isoelectric focusing?
gel is made containing a gradient of pH
in isoelectric focusing, where does protein migrate to?
isoelectric point: pH at which the protein's net charge is 0
two dimensional electrophoresis
combined isoelectric focusing and SDS page electrophoresis to achieve high resolution separation of proteins.
what does ultracentrifugation teach us about?
mas, density, shape
what is the sedimentation coefficient?
a means of quantifying the movement of a particle when subjected to a centrifugal force
s=m(1-vp)/f what are each of these coefficients
m = mass of the particle
v = partial specific volume (the reciprocal of density)
! = density of the medium
f = frictional coefficient (a measure of the shape of the particle)
(1-vp) = buoyant force exerted by the liquid medium
sedimentation coefficients are expressed in what unit?
svedberg units which is s x 10^-13
what is the relationship between s and how fast a molecule moves in a centrifugal force?
the smaller s is, the slower a molecule moves in a centrifugal field. the larger the s value, the faster a molecule moves in a centrifugal field
how does a particles mass relate to how fast it sediments?
A more massive particle sediments faster than a lighter one.
how does shape affect how fast it sediments?
Elongated particles sediment more slowly than compact,
how does density affect how fast it sediments?
A dense particle sediments faster due to less opposing buoyant
when do particles sink? when do they float? when do they not move? in terms of density?
Particles sink when vp < 1
float when vp > 1
and do not move when vp = 1
5 paramters to measure protein purification?
quantity of protein present in a fraction; determined by multiplying the protein concentration by the total volume
determined by mutiplying the activity for a
given volume of protein used in an assay by the total
total activity divided by the total protein
measure of the activity retained after each step;
the activity of the initial crude step is taken to be 100%
measure of the increase in purity after
each step; calculated by dividing the specific acitivity,
after a purification step, by the specific activity of the
ideally, how do specific activity, purification level, and yield change with each purification step?
specific activity and purification level increase while yield decreases
what is maldi-tof?
matrix assisted laser desorption ionization.
1. protein is ionized using laser pulse, acidic matrix ionizes and imparts positive charge to proteins
2. proteins accelerated towards detector using electric field
3. time of flight (tof) is measured
4. smaller molecules travel faster
how can you use malditof to identify proteins?
cleave proteins with proteolytic enzymes followed by mass spec to create a fingerprint that can be compared to other protein mass specs. also you can predict cleavage pattern from genomic DNA sequence
how would you use maldi-tof with proteolytic digests to identify proteins in a nuclear pore complex?
first use HPLC to fractionate nuclear pore complex and run it on a gel. isolate the protein band from the gel and digest it with trypsin and subject to mass spec.
from this 3 new proteins were identified
why is the amino acid sequence important?
1. similarity with a known protein could help infer function
2. comparison of the same protein in different organisms can give info about evolutionary history
3. many proteins contain signal sequences causing them to be localized to one region of the cell
what are 2 techniques to determine 3D structure of proteins?
x ray crystallography and nuclear magnetic resonance (nmr)
what is a requirement for protein crystallization:
protein must be very pure >90 percent
how is a protein crystallized?
purified protein is subject to conditions of high salt, variations in pH, addition of long polymers (polyethylene glycol) to form an ordered lattice of protein molecules
what are the 3 components of xray crystallography
protein crystal, x ray source, and an x ray detector
how does x-ray diffraction work?
when x-rays pass through protein crystals, they are deflected by the protein molecule's electron clouds, larger atoms (more electrons) will scatter xrays more efficiently than lighter atom
crystal's repeating symmetric formation will allow for deflected xrays to have constructive interference with one another
the intensity of each spot of the diffraction pattern is measured. the image of the protein is then reconstructed from this data using the Fourier transform
xray diffraction data is converted to an electron density map which indicates overall shape of molecule
a molecular model is then built to fit this map
when can you use NMR
when proteins do not readily crystallize
to see protein structures' state in a free solution
what is required for NMR
highly concentrated solutions of proteins (15 mg/ml)
what does nmr depend on
the fact that certain atomic nuclei function as small magnets