methods for identifying proteins Flashcards
what methods can be used to determine the 3D structure of a protein?
1) x-ray diffraction
2) nuclear magnetic resonance (NMR)
TRUE OR FALSE: all proteins can form crystals
false: not all proteins crystallize and some may not have a folded structure
What are the steps of x-ray crystallography?
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
A diffraction pattern spot can give us what information?
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)
What is easy to measure from diffraction matter? what will be a theoretical measure?
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.
What does an electron density map show?
where the individual atoms are in a crystal structure from x-ray crystallography
In x-ray crystallography, what is the R value?
R value is resolution and a measure of how good the theoretical diffraction pattern from the model structure fits the diffraction pattern observed experimentally
What is considered a good diffraction pattern - a low R value or a high R value?
a low R value means a better model
How can a ramachandran plot be used to assess the quality of a determined crystal structure?
- the plot can show al of the amino acids in high resolution protein structures
What are some advantages of crystallography?
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
What is nuclear magnetic resonance spectroscopy?
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
Which biological nuclei have unpaired protons ?
15N, 1H, 13C, 19F, 31P all have I = 1/2
In the presence of an external magnetic field (Bo) what spin states exist for unpaired protons?
+1/2 and -1/2
Which energy state is aligned with the external magnetic field and which is opposed to it?
low energy +1/2 = aligned
high energy -1/2 = opposed
What exactly is measured in NMR?
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
In NMR, after the individual atom has been identified how can you construct interactions model structures?
look at interactions between the nuclei due to their proximity in a dihedral0 angle or 3D space as constraints
What is 2D NMR?
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
What is 3D NMR?
coupling between 3 atoms
e.g., 15N, 13Ca, 13Cb, atoms in a polypeptide backbone
adequate for proteins smaller than about 250 amino acid residues
What is 4D NMR?
coupling between 4 atoms
What is 2D NMR COSY?
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
What is NOESY?
measures hydrogen atoms that are spatially close to one another (not necessarily bonded to one another) - critical for tertiary structure determination
How do COSY and NOESY differ?
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
Why do protons have to be w/in 6 angstrongs of each other for NOESY?
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
Planck’s equation is used for what method of protein identification/
spectroscopy
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
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
When would you use the beer-lambert law?
to measure absorbance in a spectrophotometer
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
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
What is the Beer-Lambert Law?
A=ecI, where e is the extinction coefficient, c is the concentration and I the pathlength
What is transmittance?
the ratio of intensity of transmitted light (I) to that of the incident light (Io)
The inverse of the equation for transmittance is used to measure what?
to calculate the absorbance
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
A spectrometer provides the value of what? what does it not provide?
Provides value of absorbance
does not provide transmittance
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
Peptide backbones absorb what wavelengths?
between 190 and 240nm
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
Bases absorb strongly at what region and peak where?
-absorb strongly near UV region and peak at 260 nm
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
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
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
What is a huge advantage of mass spectrometry?
you do not need a large amount of proteins, only a very small amount is required
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
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
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
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
In centrifugation, what represents the mass of the displaced solution?
volume of the solution displaced times the density of the solution
In centrifugation, when will a particle stop accelerating but remain in steady motion?
when the sedimentation force equals the frictional force
What is partial specific volume?
the change in volume when you add grams of solute with temperature and pressure being constant.
What is the experimentally determined partial specific volumes for soluble, globular proteins?
0.73
Partial molar quantities are what kind of variables?
intensive
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
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