Proteins and transcription (L1, 2, 5, 8, 9, 11) Flashcards
What forms the backbone of a protein?
Covalent bonds which link amino acids together
How many different side chains are there? Which ones are hydrophobic? Which can be phosphorylated?
20 different R groups There are 8 hydrophobic ones: - Isoleucine -Leucine - Alanine - methionine -phenylalanine - Valine - Glycine - Proline
Seirene, threonine and tyrosine can be phosphorylated
What determines the shape of a protein?
Peptides fold so the polar side chains (hydrophilic) are on the outside. They form hydrogen bonds with the water. The non-polar side chains (hydrophobic) become the core of the protein - they’re protected by the polar molecules.
Folded proteins are held together by different ionic interactions (ionic bonds, London forces, hydrogen bonds)
What is the tertiary structure of a protein?
The way in which individual secondary structural elements (alpha helix, beta sheets, random coils) pack together within a protein and between subdomains of a proteins
How is the primary structure of a protein identified?
These days, most primary structure is predicted from DNA sequence but can be obtained directly by amino acid sequencing using Edman degradation or mass spec. Often, we have an unknown protein sample, we can sample a bit of the protein by Edman degradation then search through the database to identify the rest.
How does Edman degradation work?
Used to sequence an unknown protein sample, up to 60 AAs. It is sensitive, simple and cheap.
In this method, an amino acid terminal residue is labelled and cleaved off of the peptide (without disrupting the other peptide bonds)
1. The peptide is reacted with PITC which sequentially removed AAs from the N-terminus
PITC reacts with the N-terminus AAs and creates a phenylthiocarbanyl-peptide derivative.
2. Eventually, you get separation of molecule + end AA which can be examined.
3. It takes a lot of cycles to sequence the whole protein
Prediction of the structure is based on the need for amino acids to be in certain positions to achieve a certain structure e.g. to make cross-links
What method can be used to find secondary structure?
Secondary structure can be found using circular dichromism.
It involves differential absorption of circularly polarised light. Different secondary structures give a characteristic shape of CD spectrum. (alpha gives a big downward dip, beta give a less steep dip and random coil gives an upward graph) Done using CD spectroscopy in the far UV-spectrum. CD is the differential absorption of circularly polarised light. The fraction of each type of secondary structure in a protein can be found form its far-UV CD spectrum (i.e. the fraction of alphas to betas - so can also give a bit of an indication on the tertiary structure). Also, CD signals of aromatic AAs and disulfide bonds contribute ti tertiary structure. Absorbance is affected by the local environment as well (you can observe change when heating etc)
How does Nuclear Magnetic Radiation help find the structure of proteins?
If an atom has an uneven no. of protons and neutrons its spin has a slight wobble. NMR ‘active’ atoms resonate at specific frequencies in a string magnetic field. Depending on the environment, different protons have different frequencies (chemical shift) Proteins grown in bacteria with NMR active atoms can be analysed using NMR. It is an iterative process, so you get an estimation of possible structures. Expensive and takes time. And the size of the protein is limited to approx. 50 kDa
What kind of proteins can be analysed using EM?
Large structures. EM uses negative stain of vitreous ice (cryo-EM) to preserve the specimen. Image analysis is then employed to build up an average structure. The more ordered the structure, the easier the imaging process. E.g. actin has a helical structure and viruses have radial symmetry.
Why do we need to purify proteins?
Cells contain about 2000-3000 proteins. We need to know the structure and function of all individual ones for clinical and medical research. Proteomics is the analysis of the complete number of proteins in a living system -including spliced variants and post-translationally modified proteins. Researchers may want to isolate a protein to test its potential uses e.g. as an anti-bac/ fluorescents
What does the typical protein purification protocol involve?
- tissue homogenisation (sonication, blending, grinding)
- Separation of the released material by centrifugation
- Several chromatography steps
- Confirmation of protein purity by electrophoresis, western immunoblotting or MS
What is velocity segmentation?
Centrifugation is based on density. So you can separate the sediments into layers in one spin by adding sucrose to make a density gradient.
How does gel filtration work?
Use porous beads with holes. Smaller proteins get trapped and larger ones flow through
Separation by size.
How does affinity filtration work?
Use beads with a covalently attached substrate. The target enzyme (the protein you’re looking at) will bind to the substrate. Proteins with a higher affinity will get stuck. You then elute the protein using a competitive ligand once all the others have been filtered out. The fraction with the highest activity is the purest (the majority of the proteins in it are binding)
How does ion exchange filtration work?
You use beads with a positive charge. More negatively charged molecules move through more slowly. Separation is based on charge. Increasing salt concentration is used to compete for ionic interactions. The more weakly attracted ones are slowly eluted.
Use carboxymethyl for cation exchange and anion exchange use DEAE
Why would you use all three filtration techniques together?
Using all 3 together with means you get the purest fraction. It’s very unlikely you will get multiple proteins with the same size, affinity and charge.
If you do a gel electrophoresis after you should only get 1 band if the fraction is pure.
What is an SDS PAGE?
Sodium dodecyl sulphase polyacrylamide gel electrophoresis
Used to separate proteins in terms of size in the gel. Load samples into the wall of a precast gel. polyacrylamide gels are more precise due to being more tightly knit. The proteins migrate through using an electric field towards the opositely charged electrode. Larger proteins stay nearer the loading wells because they can’t move as easily. SDS changes the charge (makes it negative) on the molecules so the amount the protein moves is proportional to the size (molecular mass) and not the charges. The proteins are heated with SDS and mercaptoethanol, so they are denatured and can be completely surrounded by the SDS negative charge.
What is a 2 dimensional gel electrophoresis and why are they used?
Used for analysing complex samples. You use isoelectric focusing as the 1st dimension and then the SDS-PAGE as the second. The isoelectric focusing separates the proteins by charge and the SDS-PAGE separates by size. The isoelectric focusing involves a gel with varying pH. The proteins stop moving at their isoelectric point. (when it has no net charge and therefore no longer migrates in the electric field)
When isn’t an SDS-PAGE a useful analysis tool?
When you’re isolating Pure samples to be used in research. Because the SDS-PAGE alters the protein, making it useful for proteomics but not if you need the intact working protein.
What is a western blot?
Also known as an immunoblot, it is a way to analyse and identify proteins. You first do an SDS-PAGE and then the proteins are electrophoretically transferred onto a membrane using an electric field (it won’t work if they’re still on the gel). Then you use immunohistochemistry (antibodies) to identify the protein. You have to use 2 antibodies (the one specific to the protein and another that with a tag that binds to that one)
How can you use Mass Spectrometry to identify proteins?
You isolate the protein then digest it using proteases like trypsin to make peptides. Then you ionise the peptides and measure their mass/charge ratio. They are also detected in proportion to their abundance. Each small peptide has a distinct mass and thus can be identified. You can also determine protein phosphorylation using MS> Out of 20, it is known that 3 can be phosphorylated (serine, threonine and tyrosine) because they have hydroxyl groups. Proteins that undergo post-translational modifications can be phosphorylated. MS has now become a critical technique for almost all proteomics as it allows for the presice determination of molecular mass as well as their sequence.
What are some post-translational modifications that some amino acids undergo?
Hydroxylation - by hydroxylases - hydroxyl proline modification of collagen leads to scurvy (+17 Da)
Methylation - methyltransferases, e.g. in histones and tubulin, adds on 15Da
Acylation - by acyltransferases - e.g. on histones - adds on about 27 Da
Lipids - palmitoylation, farnesylation. adds on 2000+ Da.
How does the structure of proteins allow ligands to bind?
Proteins fold so that they form pockets containing specific side chains. These pockets form binding sites for ligands. The ligand is usually specific for the binding site (has a similar shape - lock and key)
How do enzymes work? what is the action of some common ones?
Enzymes act by lowering the activation energy of the reaction (acts as a catalyst) Hydrolases cleave something in a hydrolysis reaction. Synthases synthesise molecules in anabolic reactions. Polymerases catalyse polymerisation reactions. Kinases add phosphates onto things. Phosphatases remove phosphates off of molecules.
What is the equilibrium constant of a protein interaction?
the equilibrium constant = conc of AB / conc of A x conc of B (look at notes for why)
What is the principle of protein-protein interactions?
The surfaces of molecules A&B and B&C are a poor match and are only capable of forming a few weak binds, therefore thermal motion would rapidly break them apart. The surfaces of A and B are a good match and therefore can form enough weak bonds to withstand thermal jolting - therefore they stay bound to each other. These bonds can be ionic, hydrophobic or electrostatic. These types of interactions require complementary surfaces. Binding enables the formation of protein complexes, like hemocyanin which is a giant oxygen transport complex (isolate from a scorpion). Binding often causes a conformational change in a protein (E.g. EF-Tu binds to GTP to become activated, eventually, the GTP hydrolyses to GDP resulting in inactivation of EF-Tu
What are the different types of surface interactions?
Surface-string, helix-helix (when helices wrap around each other), Surface-surface
Explain the binding of the SH2 domain
SHR (Src homology 2 domain) is an important phosphotyrosine binding domain often involved in signalling mechanisms. Prototypical SH@ is from tyrosine kinase Src, but it’s also found on many other signalling and adaptor proteins
Specificity is between the phosphate of the p-Tyr, mainly ionic interactions between the negative phosphate and positive amino acids, but some hydrogen bonding is also present.
Explain the binding of the SH3 domain.
The Src homology 3 domain has a poly-proline binding domain and acts as an adaptor to link proteins. It binds proline-rich motifs. SH3 has structural roles in maintaining multiprotein complexes. The minimum consensus sequence for SH3 binding is P-X-X-P. SH3 contains several aromatic residues, these interdigitate between the prolines of the PxxP motif which is stabilised by aromatic static. Binding occurs through electrostatic interactions due to aromatic stacking of Pro and Tyr.
