Protein interactions Flashcards
What are ‘pockets’ in proteins?
Binding sites for ligands - formed by the protein back bone folding to give a specific shape
How do enzymes work?
Lower the activation state of a reaction
Making it easier for the substrate to pass over the activation state to product
Reactions happen at a higher rate
What do hydrolyses do?
Catalyse a hydrolytic cleavage reaction, using water
What do nucleases do?
Break down nucleic acids by hydrolysing bonds between nucleotides
What do proteases do?
Break down proteins by hydrolysing bonds between amino acids
What do synthases do?
Synthesise molecules in anabolic reactions by condensing 2 smaller molecules to make a larger molecule
What do isomerases do?
What do they change and what do they keep the same?
Catalyse the rearrangement of bonds within a single molecule
Change the chemical structure but keep the same chemical formula
What do polymerases do?
Catalyse polymerisation reactions, such as the synthesis of DNA and RNA
What do kinases do?
Catalyse the addition of phosphate groups to molecules (especially proteins)
What do phosphatases do?
Catalyse the hydrolytic removal of a phosphate group from a molecule
What do oxidoreductases do?
Catalyse reactions in which one molecules is oxidised, whilst the other is reduced
What can oxidoreductases also be called?
Dehydrgenases
What do ATPases do?
Hydrolyse ATP
Many proteins with different functions have this as one of their functions
What is dissociation?
2 molecules bound together coming apart
What is the dissociation rate defined by?
The affinity of the molecules together and the concentration of the bound product at the start [AB]
What is the dissociation rate constant?
How is this used to calculate dissociation rate?
Koff
Dissociation rate = Koff x [AB]
What is association?
2 molecule coming together
What is association rate proportional to?
The concentration of the individual products [A][B]
What is the association rate constant?
How is this used to calculate association rate?
Kon
Association rate = Kon x [A][B]
What happens to the association and dissociation rate at equilibrium?
They are equal
Association rate = Dissociation rate
What is the equilibrium constant and what is it equal to?
K
K = Association rate = Dissociation rate K = Kon [A][B] = Koff [AB] K = Kon/ Koff = [AB] / [A][B]
How can association be measured?
1) Hold A constant
2) Gradually increase B in solution
3) Measure the fraction of bound and unbound ([AB] / [A] )
4) Overtime AB increase until SATURATION
What is the shape of an association curve and why?
- Sharp increase in the fraction bound ( [AB] / [A] ), when B is first added
- Then a slower gradual increase, as the molecules are continuously dissociating
- Eventually, graph flattens out as the reaction is saturated
What are ‘transient interactions’?
Interactions between proteins - they have a dissociation constant and don’t stay bound
Why must the shapes of proteins be compatible?
To from many bonds (ionic, hydrophobic, electrostatic)
What are 3 types of protein interaction? (Describe them)
1) Surface-string (string is the ligand, fits into a groove)
2) Helix-helix (2 strings wrapped around each other)
3) Surface-surface (complimentary surfaces)
What does binding between proteins often cause? (2 things)
A conformational change in shape
A formation of a protein complex
What do signalling pathways rely on?
Interactions between a few conserved domains
Eg.
- Receptors
- Intracellular proteins which transduce the signal
What are protein domains?
Modular parts of a protein which have specific purposes and can evolve, function and exist independently of the other proteins in the protein chain
What happens to protein domains during evolution?
What does this form?
They are swapped with each other - forming many different functional domains which have taken advantage of the specific domains
Forms many different proteins, which can all have the same domain, but are paired with other modular domains
What are 6 examples of commonly occurring (conserved) domains?
What do they bind?
ALL bind specific substrates
1) SH2
Binds phosphotylated tyrosine
2) SH3
Bind proline-rich motifs (POLY-prolines)
3) PH
Bind phospholipids
4) EF hand
Binds calcium/magnesium in structural of signalling roles
5) Zinc finger
Bind zinc in a structural mode
6) Leucine zipper
Mediated protein-protein or protein-DNA binding
Where were the SH2 and SH3 domains originally found?
What is the role of this protein?
In Src
Important in cancer - researchers wanted to know what each domain did
Where is SH2 domain found?
In MANY unrelated different proteins
What is the SH2 domain involved in and how?
Signalling mechanisms:
- Binds to phospho-tyrosine ligands to form signalling complexes (docking)
What can increase binding to SH2?
Kinases - by phosphorylating tyrosine
What can decrease binding to SH2?
Phosphatases - by removing the phosphate from tyrosine
How does the SH2 domain bind to phospho-tyrosine?
Has a binding pocket, which is lined with positively charged amino acids
(phosphate on the tyrosine is NEGATIVELY charged)
What does the SH3 domain do?
What is it involved in?
Act as an adaptor to LINK proteins, bringing them together in a non-regulated fashion
Involved in signalling and has a structural role (holds proteins together - maintaining a mul-tiprotein complex)
What is the minimum consensus of a target of SH3?
P-x-x-P
with x being another amino acid
How does the SH3 domain interact with the target?
NOT through phosphorylated tyrosine!!
SH3 domain contains many AROMATIC residues, which interdigitate between the prolines of the PxxP motif
Stabilised through AROMATIC STACKING (as proline is also aromatic)
What is ‘aromatic sticking’?
Non-covalent interactions between aromatic rings
Causing stabilisation of the aromatic side chains into a LOW ENERGY state
What is the difference between the interactions of SH2 to its target and SH3 to its target?
SH2 - mostly IONIC bonding (H bonding or charged attraction)
SH3 - ELECTROSTATIC interactions (Van Der Waals)
What is the PH domain involved in and how?
Signalling and anchoring proteins to the membrane
As binds to phosphorylated lipids
How does the PH domain bind to different lipids?
Lipids show different states/levels of phosphorylation, which is recognised by the PH domain
Where does the PH domain sit in the cell and why?
Close to the membrane - binds to phosphorylated lipids (in the membrane)
What creates binding sites for proteins containing PH domains?
Kinases - phosphorylates lipids
What pathways are PH domains involved in? (5 things)
1) Actin rearrangement
2) Vesicular trafficking
3) Gene expression
4) Cell growth
5) Survival
What is Spectrin?
A structural protein with a PH domain
How does the PH domain in Spectrin bind to the target?
Has amino acids to make the binding site ‘hydrophillic’
Pocket is +ve charged to interact with the -ve phospholipids
To make the protein ‘comfortable’ against the hydrophilic heads of the lipids in the membrane
What function do Zn ions have in the cell?
Structural - holds proteins in a certain confirmation
What function do Ca ions have in the cell?
Regulatory - Tightly controlled concentrations within the cell
What function do iron and copper ions have in the cell?
Enzymatic - found within enzymes
What do EF hands do?
Bind Ca ions to have a regulatory function
Bind Ca or Mg ions to have a strutural function
What does calcium binding to EF hands cause?
Induces a structural change
How does the size or valency (charge) of a metal ion change its binding?
Different sizes and valencies of metal ions need to be liganded by different numbers of amino acids and have DIFFERENT structures
What is the structure of an EF hand?
Octadentate (has 8 atoms in the binding site which can interact with the calcium ion)
7 oxygen containing side chains
Invarient glycine residue to accommodate for the oxygen which encircles the calcium - tight turns
2 alpha helices
What purpose does the oxygen in the side chains in the EF hand serve?
They are negatively charged - to shield the positively charged calcium ion, which is embedded tightly in a pocket with a bed of oxygen
What is calmodulin?
A regulatory protein which is involved in many signalling pathways
Commonly binds to calcium
What is the structure of calmodulin?
2 binding domains with 2 EF hands - can bind to 4 calcium in total (2 on either side)
Glycine - to allow the sharp bend
What happens when calcium binds to calmodulin?
What does this allow?
Conformational change to expose a hydrophobic patch of amino acids on each EF hand
Allows the interaction with amphipathic alpha helices (have BOTH hydrophobic and hydrophilic parts)
Why do proteins need to interact with DNA?
To regulate:
- DNA activity
- Gene expression
- DNA replication
- RNA synthesis
How are DNA binding domains similar?
- Overall basic charge (have basic amino acids) as DNA is acidic
- Recognises DNA strand mostly through interactions with the major groove (wider - easier for amino acids to interact)
What are 4 examples of DNA binding motifis, which are present in DNA binding proteins?
1) Zinc fingers
2) Leucine zippers
3) Basic helix-loop-helix (bHLH)
4) Beta-sheet
Why do many DNA binding proteins have several DNA binding motifs in a row?
For increased affinity and a more stable and specific interaction
Interact with multiple places on the DNA
What do zinc finger bind to?
DNA
Zn
How and why do zinc fingers bind to Zn?
To stabilise the DNA binding structure - holds the alpha helices and beta sheet together in the tertiary structure
4 amino acids (histidine and/or cystine) separated by at least 2 residues bind to the Zn ion
Zn ions are also positively charged - DNA is negatively charged
Which part of the Zinc finger interacts with the DNA and how?
The alpha helice, interacts with the major groove
What is proline?
An amino acid
