methods for identifying proteins Flashcards
(133 cards)
1
Q
what methods can be used to determine the 3D structure of a protein?
A
1) x-ray diffraction
2) nuclear magnetic resonance (NMR)
2
Q
TRUE OR FALSE: all proteins can form crystals
A
false: not all proteins crystallize and some may not have a folded structure
3
Q
What are the steps of x-ray crystallography?
A
1) start with protein crystal
2) take it to an x-ray source to obtain diffraction pattern
3) use diffraction pattern and fourier transform to generate an electron density map
4) interpret density map into 3D structure - can represent functional conformation
4
Q
A diffraction pattern spot can give us what information?
A
1) Amplitude of scattered beam (F. value) - how bright the scattered beam is at each spot relative to F value or amplitude of scattered wave. different spots have different F values
2) Phase φ values - phase of scattered bean - location of scattered beams related to how the peaks and through align in space to give rise to the location of spots ( where in 3D space reflected beams give rise to signals vs. locations where there is no signal)
5
Q
What is easy to measure from diffraction matter? what will be a theoretical measure?
A
F values is easy to measure and phase value needs to be constructed through structure models of the scattered data
-with this you can measure electron density and mapp it out.
6
Q
What does an electron density map show?
A
where the individual atoms are in a crystal structure from x-ray crystallography
7
Q
In x-ray crystallography, what is the R value?
A
R value is resolution and a measure of how good the theoretical diffraction pattern from the model structure fits the diffraction pattern observed experimentally
8
Q
What is considered a good diffraction pattern - a low R value or a high R value?
A
a low R value means a better model
9
Q
How can a ramachandran plot be used to assess the quality of a determined crystal structure?
A
- the plot can show al of the amino acids in high resolution protein structures
10
Q
What are some advantages of crystallography?
A
1) knowledge of accurate molecular structures can help with rational drug design and for structure-based functional studies to aid development of effective therapeutic agents and drugs
2) crystallography can reliably provide the answer to many structure-related questions, from global folds to atomic details of bonding
3) no size limitation exist for the molecule to be studies
4) 2-way impact on/by genetic engineering, computer science, synchrotron protein engineering
11
Q
What is nuclear magnetic resonance spectroscopy?
A
NMR uses radio frequency waves to disturb the spin state of an UNPAIRED proton in a nucleus that is under the influence of a strong external magnetic field
12
Q
Which biological nuclei have unpaired protons ?
A
15N, 1H, 13C, 19F, 31P all have I = 1/2
13
Q
In the presence of an external magnetic field (Bo) what spin states exist for unpaired protons?
A
+1/2 and -1/2
14
Q
Which energy state is aligned with the external magnetic field and which is opposed to it?
A
low energy +1/2 = aligned
high energy -1/2 = opposed
15
Q
What exactly is measured in NMR?
A
the aligned spins for different nuclei are disturbed and their relaxation back to the initial state is measured
-nuclei in different chemical environments will have different relaxations dynamics and this can be measured to identify individual atoms in the structure
16
Q
In NMR, after the individual atom has been identified how can you construct interactions model structures?
A
look at interactions between the nuclei due to their proximity in a dihedral0 angle or 3D space as constraints
17
Q
What is 2D NMR?
A
2D is the coupling between atoms, e.g, H atoms with other H atoms or H atoms with C atoms
adequate for proteins smaller than about 100 amino acid residues
18
Q
What is 3D NMR?
A
coupling between 3 atoms
e.g., 15N, 13Ca, 13Cb, atoms in a polypeptide backbone
adequate for proteins smaller than about 250 amino acid residues
19
Q
What is 4D NMR?
A
coupling between 4 atoms
20
Q
What is 2D NMR COSY?
A
Correlation spectroscopy.
there are two radio pulses turned to perturb specific NMR active nuclei. In COSY, multiple time dependent signals are measured as the time between the pulses is varied. This will give a 2D frequency spectrum at fourier transformation
AKA COSY MEASURES ATOMS THAT ARE BONDED TO ONE ANOTHER
21
Q
What is NOESY?
A
measures hydrogen atoms that are spatially close to one another (not necessarily bonded to one another) - critical for tertiary structure determination
22
Q
How do COSY and NOESY differ?
A
cosy measures nuclear spins interacting through chemical bonds or bond coupling and noesy measures how nuclear spins influence each other through space, usually if they are within 6 angstroms of one another
23
Q
Why do protons have to be w/in 6 angstrongs of each other for NOESY?
A
2 protons closer than 5 angstrons have coupled spins even if they are not close in the primary structure ( the through-space effect)
thus, NOESY shows only protons that are close in the 3D structure but not necessarily in the primary sequences
24
Q
Planck’s equation is used for what method of protein identification/
A
spectroscopy
25
What does planck's equation tell us?
it gives us the energy of a photon, where the energy E = the product of the planck's constant and the frequency of light.
The frequency is speed of light divided by wavelength
26
How are energy and wavelength related?
energy is inversely proportional to the wavelength
SO, if the wavelength is really long, the energy is low
if the wavelength is short, the energy is high
27
When would you use the beer-lambert law?
to measure absorbance in a spectrophotometer
28
What does a spectrophotometer measure?
light absorption, which is then used to detect and identify the compound of interest or to calculate the concentration of the compound in a solution
29
In a spectrophotometer, what does the cuvette contain?
c moles/litres of the absorbing molecule
The fraction of the incident light absorbed by the molecule is related to the thickness of the absorbing layer or the path length (which is the width of the cuvetter) and the concentration of the absorbing species = this is quantified by the beer lambert law
30
What is the Beer-Lambert Law?
A=ecI, where e is the extinction coefficient, c is the concentration and I the pathlength
31
What is transmittance?
the ratio of intensity of transmitted light (I) to that of the incident light (Io)
32
The inverse of the equation for transmittance is used to measure what?
to calculate the absorbance
33
What is another name for extinction coefficient and what does it measure ?
used in spectrometry and measures how strongly a particular chemical species absorbs light at a given wavelength
extinction coefficient is also called molar absorbtivity
34
A spectrometer provides the value of what? what does it not provide?
Provides value of absorbance
does not provide transmittance
35
Which 4 amino acids absorb at 280nm? what can this knowledge be used for?
Tryptophan and Tyrosine
- most proteins have Tyr and Trp in primary sequence, Tyr being more common. This property can be used to characterize the protein
- Phenylalanine and Cystine also absorb at 280nm
36
Peptide backbones absorb what wavelengths?
between 190 and 240nm
37
How does cystine contribute to molar absorptivity?
it can absorb wavelengths of 280nm but also when 2 cysteine molecules come together they form a disulfide bond
38
Bases absorb strongly at what region and peak where?
-absorb strongly near UV region and peak at 260 nm
39
How do we find if DNA is pure using absorbance ratio?
use ration of 260nm/280nm =1.7 to 2.0 is considered pure for DNA, anything greater than 1.7 is considered pure
40
What is the hypochromic effect?
when ssDNA, dsDNA, RNA and isolated or free nucleotides have different absorbance due to structural properties of the nucleic acids. The stacking of bases in the core of the double helix decreases absorbance. So single stranded DNA has a higher absorbance than double stranded DNA and at 260 nm the unfolding of DNA can be monitored due increase in absorbance from dsDNA to ssDNA
41
What information can mass spectrometry give?
- used for protein characterization and identification
- it can analyze ionized forms of molecules in the gas phase
- it can determine mass of protein or nucleic acid
- provide amino acid sequence of short polypeptides
42
What is a huge advantage of mass spectrometry?
you do not need a large amount of proteins, only a very small amount is required
43
How does mass spec determine the mass of a particel?
by how quickly the ion accelerates under an applied electric field
- lighter ions move faster
- if we can measure acceleration, we can determine the mass.
Acceleration = mass/charge
Force = mass times acceleration
44
What are the different types of mass spectrometry ?
1) Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
- protein is mixed w/organic compound that can absorb light of an appropriate wavelength
2) Electrospray ionization mass spectrometry (ESI MS)
- macromolecules are forcibly converted from liquid to gas phase by electrospray
3) Tandem mass spectrometry (MS-MS)
- two mass specs are used in tandem - this method provides the mass of the macromolecule but also the sequence
45
What do centrifuge do and how do they separate molecules?
separates molecules based on size, shape, and density
centrifuge spins samples at high rotational speeds and generates centrifugal force
46
What is the sedimentation force comprised of?
two opposing forces, the centrifugal force and the buoyant force
sedimentation is the difference between the centrifugal force and the buoyant force
47
In centrifugation, what represents the mass of the displaced solution?
volume of the solution displaced times the density of the solution
48
In centrifugation, when will a particle stop accelerating but remain in steady motion?
when the sedimentation force equals the frictional force
49
What is partial specific volume?
the change in volume when you add grams of solute with temperature and pressure being constant.
50
What is the experimentally determined partial specific volumes for soluble, globular proteins?
0.73
51
Partial molar quantities are what kind of variables?
intensive
52
TRUE OR FALSE: The sedimentation constant is proportional to the mass of the particle, which is the molecular weight divided by avogadro's number
TRUE
53
Mass, friction and sedimentation have a relationship in centrifugation, what is it?
mass and S have a proportional relationship as mass increases, sedimentation constant increases
Frictional force and sedimentation have a inversely proportional relationship as friction increases, S decreases
54
How is the frictional coefficient determined?
Using stokes equation, which assumes the particle is spherical and unsolvated. IF the particle is a sphere, you take radius into account
55
Is the frictional coefficient higher or lower for a non-spherical particle vs a spherical one
higher for a non-spherical than for a spherical.
for non spherical the f value is higher than 1.5 for a spherical it is between 1.1 and 1.5
56
Which centrifugation methods use density gradients?
1) sedimentation velocity, aka zonal - uses pre-made gradient (e.g., sucrose)
2) sedimentation equilibrium aka isopycnic (uses gradient made during centrifugation, e.g., CsCl
57
In the sedimentation velocity centrifugation, separation of particle is based on what?
based on the S value, therefore the mass and shape of the particle, similar shapes move according to mass
there is a a linear dependence between the sedimentation rate and the s value where sedimentation rate is how fast the particle has traveled through the tube or sample
58
For sedimentation velocity, why must one ensure the density of the gradient is less than the macromolecule of interest?
b/c as you spin the macromolecules in the density gradient move in zones
-if the macromolecule was lighter it would stay at the top of the tube never settling to the bottom
59
What is the sedimentation rate?
how fast the particle has traveled through the tube or sample.
60
in sedimentation velocity, what happens if you spin for too long?
everything pellets aka reaches the bottom of the tube
61
How do components in a centrifuge tube move towards the bottom in relation to their S (sedimentation value)
- High s value = have a higher sedimentation rate = lower on the column
- Low S value = low sedimentation rate = higher on the column
62
How do you collect fractions from a centrifuge?
you make a hole in bottom of the tube and collect them as they come off
63
Would a molecule that is smaller and less compact have a lower or higher s value?
-low S value - stay at top of the tube
64
Would a molecule that is bigger and more compact have a higher or lower s value
-high s value go to bottom of tube
65
How is sedimentation velocity different than sedimentation equilibrium (isopycnic centrifugation)?
sedimentation velocity does not separate on equilibrium and separates based on mass and shape, gradient is also pre made. where as sedimentation equilibrium separates only on density - where a particle moves down the gradient till it reaches a density that matches its own. and the gradient is established during the centrifugation
66
in sedimentation equilibrium, what happens after a particle reaches a density that matches its own?
it will stop there and not travel any further even if. you continue to spin.
67
If you have proteins, which method (sedimentation velocity vs. sedimentation equilibrium) would you use ?
use sedimentation velocity over sedimentation equilibrium b/c proteins have a uniform density and since sedimentation equilibrium is based on density, the proteins would not separate from each other.
68
Sedimentation equilibrium is best for what types of molecules?
membranes, nucleic acids but not proteins
69
What is a common dense gradient forming material used for sedimentation equilibrium?
CsCl
70
What are the steps in sedimentation equilibrium?
1) Mix nucleic acid or sample with dense gradient forming material such as CsCl
2) let this spin till nucleic acid molecules reach a position that is equal to their buoyant density
3) Remove sample band via a hypodermic needle and suck up sample from column
4) visualize by mixing in EtBr
71
What are two methods for analytical centrifugation?
1) kinetic - gives s value
| 2) equilibrium - gives molecular weight
72
Since kinetic and equilibrium analytical ultracentrifugation is non preparative what does this mean about the samples?
1) you will use small volumes of sample
| 2) done in physiological buffers
73
What does analytical centrifugation (kinetic and equilibrium) monitor?
monitors absorbance while spinning by using cuvettes in the rotor and a light source and detector
74
What does frictional force depend on?
size
shape
solvation
viscosity
75
What is the general principle behind elecrophoresis?
in an electric field, charged particles migrate towards the pole of opposite charge.
Particle accelerates based on electric field force and encounters an opposing frictional force, when they equal each other, there is no more acceleration but a constant velocity instead
76
What does frictional coefficient depend on?
primarily size and shape of the charged molecule but some dependence on solvation and viscosity
77
Electrophoretic mobility is directly proportional to what and inversely proportional to what?
Based on this, how would a large and less compact molecules will move?
Electrophoretic mobility is proportional to charge and velocity aka as charge increases so does mobility
electrophoretic mobility is inversely proportional to frictional coefficient and electric field aka as they increase mobility decreases
-large and less compact molecule would face more friction and thus move more slowly
smaller molecule would face less friction and move more rapidly
78
What is meant by poly-anionic?
molecules that are already charged like DNA and RNA (except at low pH)
79
How do we give charge to an uncharged molecule?
Coat proteins with SDS sodium dodecyl sulfate which is a negatively charged detergent and gives protein an uniform negative charge independent of sequence or pH
80
What are the common gel matrices used in electrophoresis?
1) Agarose gels
| 2) polyacrylamide gels
81
What are agarose gels? What can it separate?
made of seaweed and used for electrophoresis it is a looser matrix used to separate large molecules like chromosomes, RNA, DNA and protein-nucleic acid complexes
82
What is a polyacrylamide gel? What thee components are necessary for this gel ?
polymers of the molecule acrylamide that are cross-linked with 1) bis-acrylamide. for polymerization to occur, the bis-acrylamide is critical.
2) ammonium persulfate is also necessary as it forms free radicals that will initiate polymerization proces
3) TEMED is the catalyst that speeds up the reaction
83
WHat determines pore size in polyacrylamide gel?
the ratio of bis-acrylamide to acrylamide and the total concentration of both components
84
What is a discontinous buffer system? What is another name for this?
- when there is more than one gel with different pHs
- two gels: stacking gel (larger pore size ) and resolving gels
other name: Laemmli gels
85
What is a continuous buffer system?
same buffer at constant pH is used in the sample, gel, and chamber
86
When is it ideal to use a continuous buffer system vs. a discontinous?
highly concentrated proteins = continuous system
dilute protein samples = discontinuous system
87
How does glycine behave in a discontinuous buffer system?
It can exist in a positive, neutral, and negative charged state - controlling the state of glycine is key to this buffer system
1) pH 8.3 at running buffer, glycine is negatively charged so when power is turned on the negatively charged glycine ions enter the stacking gel where pH is 6.8
2) pH 6.8 glycine switches predominantly to the zwitterionic state (neutral charge) - this loss of charge causes glycine to move more slowly in the electric field compared to Cl- ions
the proteins in the gel sample will have an electrophoretic mobility that is between the mobility of glycine and Cl- causing proteins to be concentrated into a narrow zone between the Cl- and glycine front
3) pH 8.8 running gel at bottom. Glycine are mostly negatively charged and can migrate much faster than the proteins
as a result, proteins are lined up as a very narrow band at the interface of the stacking and running gels and since the running gel has an increased acrylamide concentration, aka smaller pore size, the proteins will separate according to their size.
88
What would happen if we have a large amount of protein samples and try to run it on a continous gel system?
in the absence of a stacking gel, the sample would sit on top of the running gel and rather than being lined up together and hitting the running gel together this would mean the protein would all enter the running gel at different times, resulting in a very smeared bands.
aka stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands
89
How does SDS swamp out amino acids with a negative charge? SDS reduces disulfides to what?
1) for every 2 amino acids one SDS page is used to swamp out intrinsic protein charge
2) disulfides S-S --> 2 sulfhydryls 2-SH
90
What does SDS normalize on proteins?
normalizes mass to charge ration. Denatures so minimizes shape effect, reduces disulfides too and prevents internal formation so anomalous migration is possible.
91
What are the applicaitons of SDS?
check protein purity and estimate molecular weight
92
When would you want to use native page over sds page?
when you need a protein that is not denatured and use a discontinous buffer system w/no reducing agent keeping protein in its native state
93
In native page, what affects the molecules electrophoretic mobility? How do compact globular/spherical proteins moved as opposed to elongated or rod shaped ?
size, shape and charge
globular - will run faster than elongated or rod shaped b/c frictional coefficient is smaller for spherical than rod shaped - but charge can also affect mobility so there will be a size to charge ratio
94
Give an example of how proteins can have same size different charge in native page
Lysozyme and myoglobin have the same size but the pIs (isoelectric focusing point) are different. Hence at a given pH they will have a different charge and they will run differently on a native gel.
95
WHat are the applications of native page?
1) assay the protein directly on the gel itself (without having to cover its inherent charge like in sds page)
2) utilize native page to identify proteins complexes and separate proteins of similar size but different charge
96
In electrophoresis, once you have separated bands, how can they be visualized?
1) coomassie staining (R type)
2) silver staining
3) sequence from gel (Edman degradation and MALDI-MS)
4) autoradiogram
5) immunoblot (western blot)
97
What is isoelectric focusing?
a technique for separating different molecules by differences in their isoelectric point (pI)
it moves in gradient until pH = pI
98
What is isoelectric focusing useful for?
purification, homogeneity, isoforms, post translational modification
99
What is 2D electrophoresis?
combines isoelectric focusing and SDS-page
1) it will first separate proteins w/in a similar molecular weight but different pIs
2) then separates proteins with a similar pIs but different molecular weight
100
What is capillary electrophoresis?
Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius
-gives very fast separation, high resolution, and only needs small amounts
101
What can capillary electrophoresis detect?
1) trace compounds
2) analysis of complex mixtures of small molecules
3) metabolomics: identifying metabolites in a complex mixture such as a living cell
102
What are the two phases in chromatography?
1) mobile phase - moves through the stationary phase picking up compounds to be tested usually a liquid or gas
2) stationary phase - phase does not move and has pores and is solid for mobile phase to move through
103
What is chromatography?
separates proteins on properties such as size, charge, or binding affinity
104
What is the general procedure of chromatography?
1) apply complex mixture in mobile phase
2) alter solvent conditions to change preference between phases
3) components become fractioned
3) separation downs length of column
105
Paper and thin layer chromatography is used to separate what?
small compounds - amino acids, peptides, sugars, nucleotides, lipids, other small organic compounds
106
In paper and think layer chromatography, what is the stationary phase and what is the mobile phase?
Paper:
stationary - paper, which is polar
mobile - solvent (hydrophobic, non polar)
Thin Layer:
stationary - silica on a glass plate
mobile - organic solvent
107
What can paper chromatography detect?
- color pigments, fluorescence, ninhydrin (amino acids)
| - carotenoids and chlorophil
108
What is fluorescence quenching?
a component of thin layer chromatography where inorganic phosphorus is mixed w/the adsorbent before it is coated on the plate. helps tthe plate fluoresce when exposed to UV
109
What are the types of resin in liquid chromatography?
1) ion exchange chromatography - separates on charge
2) reverse phase and hydrophobic interaction chromatography - separates based on polarity/hydrophobicity
3) affinity chromatography - separates on binding
4) gel filtration - separates on size
110
What are the types of liquid chromatography?
1) HPLC (high-pressure LC) - uses 800 psi to separate proteins
2) FPLC (fast protein LC) uses high pressure but not as high as HPLC
3) conventional - uses ambient pressure
111
what is a pro/con of HPLC?
pro: very high pressure creates high resolution
con: can only use small volumes of proteins, resin has to be highly specialized aka more expensive technique
112
What is pro/con of FPLC
pro: since pressure is not as high as HPLC, can increase capacity of volume and still get good resolution
con: resolution not as great as HPLC
113
Pro/cons of conventional LC
pro: cheaper allows large scale
con: slower, less resolution
114
What is the general set up for liquid chromatography?
1) equilibrate resin
2) load sample
3) wash
4) elute using gradient
5) search elute
115
What are two methods used to detect /identify protein of interest in chromatography
1) assay by size/sequence
| 2) assay by function
116
How can you detect protein by size after you have isolated your protein using chromatogrpahy?
SDS-PAGE
| -excellent for assessing purity. For this method one already knows the molecular weight of protein of interest.
117
How can you detect proteing by sequence after you have isolated using chromatography ?
Antibody must be present - ELISA or Western Blot
118
How can you detect protein by function after using chromatography?
Enzymatic assay or binding assay
for binding assay: EMSA (electrophoretic mobility shift assay) and filter binding w/radioactive ligand
119
What is ion exchange liquid chromatography?
utilizes the difference in sign and magnitude of the net charge on the protein at a given pH
120
What makes a weak ion exchanger?
groups that titrate with pH
121
What makes a strong ion exchanger?
groups that have a fixed positive or negative charge
122
Anion exchange chromatography uses what type of resin? what range of pI must proteins be?
cationic resin (positive charge)
uses with proteins that have a low pI (lots of D and E)
123
for anion exchange chromatogrphay, what are strong and weak ion exchangers?
strong: diethylaminoethyl (DEAE)
weak: quaternary amine (MonoQ)
124
For cationic exchange chromatography, what type of resin is used? what is the pI range for proteins to use for this?
anionic resin (negative charge)
use w/proteins that have a high pI (lots of K, R)
125
For cation-exchange chromatography, what is a weak ion exchanger and what is a strong ion exchanger?
weak: carboxymethyl (CM) and phosphate
strong: methyl sulfonate (MonoS)
126
If the resin is positively charged, (cationic column) how will proteins with a positive charge move?
Positive charge will migrate more rapidly siince it won't be attracted to column. Negative charge migrate slowly
127
When should anion exchange be used over cation exchange chromatography ?
if your protein contains lots of Asp, Glu then it is an acidic protein and has a pI < 7, at pH 7 these residues are negatively charged and you would want to use a positive charged resin aka you use an anionic chromatography with a cationic resin
128
What is the elution gradient in reverse phase liquid chromatography?
polar to non-polar, it is denaturing the protein but excellent for peptides
129
What is the general principle for affinity liquid chromatography?
based on ligand binding so you need a ligand
will elute with salt
130
In size-exclusion chromatography, how do large proteins elute vs smaller ones?
large proteins elute before smaller ones
131
What does isocratic elution mean?
there is no gradient or change in elution buffer
132
What are some thing to consider when carrying out size exclusion chromatography ?
One is if the protein interacts with the resin, there would be unintended retardation. This can then affect the elution time or elution volume. Normally we would use KCl or NaCl to prevent the protein from interacting with the resin.
The next consideration is the sample volume, that is how big or small of sample should one use relative to the total volume. In general sample volume should be 1‐2% of total volume. This allows you to get enough resolution of your protein sample. The analytical applications include determining the oligomeric state of the protein and protein –protein interactions. E.g., You have a protein that is 20 kDa and another protein which is 40 kDa. If these two proteins interact, then they will elute at 60 kDa.
133
What is dialysis?
You have a semipermeable membrane, e.g., cellulose acetate, containing a concentrated solution of the protein. The larger blue molecules represent proteins, DNA or RNA, while the buffer or salt molecules are in red. If you have a larger concentration of salts within the membrane, then the salt will travel from inside to the outside the membrane until the salt concentrations on each side are equal. Dialysis membranes have different MW cut offs, so you would select the membrane appropriate for your protein of interest.
