How are Proteins Studied? Flashcards
Previously, in order to study proteins, they had to be puryfied from the source i.e. tissue -> why was that disadvantagous? What general improvements made this easier?
Disadvantages:
- need for fresh tissue (probably had to go to slaughter houses early in the morning)
- complexity of the tissue -> takes way more time to get the protein of interest
- we only get few miligrams
Now:
- we use cultures of cells genetically modified to produce more of the protein
How does the purification procedure work?
1) Break open the cell -> releases content = cell homogenate/extract
2) Initial fractionation = process of separating targets from the rest of the molecules e.g. all soluable proteins
- now we need THE protein
3) Series of chromatography steps = use of different materials to separate based on individual properties of the protein e.g. size, shape, electrical charge
-> after each separation steps “fragments” are examined for the protein -> additional chromatography if needed
4) Once dissociated from the mixture, the protein can be further studied for its properties
NOTE: chromatography - “chroma” = color, “graphe” = writing
What is the affinity chromatography?
= form of chromatography that separates polypeptides on the bases of their ability to bind to a specific molecule
- e.g. antibodies that recognize the protein could be attached to the matrix of chromatography and used to extract it
- we may also extract the molecules that physically interact with the protein of interest
What is electrophoresis?
= technique in which proteins are loaded into a polymer gel and exposed to an electric field -> depending on their size and electrical charge they will differ in the speed at which they migrate through the polymer gel => can be stained and viasualized
What technique can be used for determining the amino acid sequence of a protein (i.e. its structure)?
Mass spectrometry = determines the exact mass of every peptide fragment within a protein that can be then compared to a database containg the list of proteins encoded by the organisms (can be computed if we know the genetic code of the organism)
How does mass spectrometry work?
1) Peptides are blasted with a laser
2) Heating up of the peptides -> become electrically charged and ejected in the form of a gas
3) Applying electrical field -> ions will fly towards a detector
4) Detector takes notice of the time it arrives
- which depends on its mass and charge
- the larger -> the slower, the more charged -> the faster
=> information gathered works as a fingerprint of the protein
NOTE: if we need to conduct the analysis on more complex mixtures of proteins -> we tend to use Tandem mass spectrometry (after passing through the first spectrometer -> broken down into even smaller fragments)
How would we predict the protein’s 3D conformation?
X-ray crystallography
- X-rays are for of elctromagnetic radiation that is shorter in wavelength than light -> we can probe extremely small objects at the atomic level
- before it can be applied the protein must be crystalized
- send a narrow beam of X-ray wave -> waves refracted and scattered by the atom -> some cancel, some remain -> electric detectors -> compute based on intensity and position of the signal
-> BUT not all proteins can crystalize
Nuclear magnetic resonance (NMR) spectroscopy
- works similarly to MRI -> nuclei of atoms align to a large magnet -> disrupted with a radiowave -> as they return back they release energy that is then detected
NOTE: Only on stationary proteins (dynamic changes cannot be visualized)
How did genetic engineering help us in this field?
1) To capture specific cells that can mass produce a certain protein
- for research
- for clinical practice e.g. insulin, fertility-enhancing drugs
2) To produce new proteins and enzymes for specific tasks
- e.g. synthesizing drugs, metabolizing toxic waste
- most not us effective as actual enzymes but we’re slowly getting there
Breaking cells and tissues is the first step to purification of proteins -> how is it done (dif ways)? See what is the most important:
1) Cell lysis (breaking cells open)
2) Cells can be lysed mechanically (high-frequency sound, pressure) or chemically (detergents).
Next fragmentization is done-> What do we use for it ?
- Note: bigger or heavier objects will end up at the bottom of the centrifuge
What do we mean by differential centrifugation?
Repeated centrifugation of higher speed can separate progressively smaller objects
How come the substances don’t mix again after the centrifugation took place?
Look at how equilibrium sedimentation works:
Look at the column chromatography:
What kind matrices can be used for column chromatography?
We can separate proteins based on their characteristics (size, charge, affinity for a substrate, etc.).
Look at the works of Gel Electrophoresis:
What do we mean by Isoelectric focusing?
Look at the 2-dimensional polyacrylamide-gel electrophoresis:;
