L5: Protein Interactions Flashcards
What can proteins interact with?
- Other proteins - hormones, receptors, subunits in protein assembly, cell-surface proteins
- Small ligands - metabolites, metal ions, neurotransmitters, antigenic peptide
- DNA/RNA
- Membranes - lipids
- Sugars/carbohydrates
How do proteins interact with other proteins or DNA or lipids?
- Using mostly non-covalent interactions - ionic interaction, hydrogen/electrostatic, hydrophobic and van der Waals
- Rarely two proteins can be joined with covalent disulphide bonds (S-S)
How does the number of protein interactions affect the strength of the overall interaction?
The more interactions, the stronger the bonding
- On their own the interactions are weaker but combined are very strong
- A small number of interactions is still unfavourable to a binding interaction with more interactions as this is stronger
What is an example of a protein interacting with a small ligand?
Calmodulin with Ca2+ ions
Describe the interaction of calmodulin with Ca2+
- Calmodulin is the main sensor of Ca2+
- When calmodulin binds to Ca2+ the calcium changes conformationally, allowing the binding of other targets - can interact and regulate the behaviour with other membrane transport proteins and a range of enzymes, and allow signalling so calcium can interact with other molecules
What are multimeric proteins?
When proteins interact and bind with other proteins of the same subunit or very similar subunits; to form a larger protein complex
What are examples of multimeric proteins?
- Structural filaments like microtubules, actin filaments and microfilaments found in cells
- Form very rigid structures that provide shape and stability to cells
What is an example of a large protein complex formed from protein-protein interactions?
- Helicase in DNA replication
- Allow the unwinding of the DNA double helix to form the replication fork
How is DNA organised in the nucleus?
- Arranged as chromatin, which is associated with histone proteins - subunit is a nucleosome
- One nucleosome is made of an octameric core with 146 nucleotides wrapped around one histone
- These structures are all connected and are further tightly wound into centromeres which make up a chromosome
- All dependent on DNA protein interactions
What are the 2 types of DNA interaction?
- DNA sequence non-specific - proteins that can interact with any DNA e.g. histones, helicases, polymerases, transcription factors
- DNA sequence specific - restriction enzymes, transcription factors - recognise very specific sequence of nucleotides and cannot bind to others
Describe protein-lipid associations
- Some integral membrane proteins which anchor and pass through the lipid bilayer. These can have hydrophobic centres that pass through the hydrophobic lipid bilayer
- Some associate and attach extrinsically through the modification of membrane proteins, to act as receptors in cell signalling - these are glycolipids
Describe protein-sugar associations
- Often seen as glycoproteins on the outside of the phospholipid bilayer
- These are proteins modified with sugar/carbohydrates
- Allow interaction and cell signalling with the external matrix
What factors determine proteins interacting with other proteins/molecules?
- Availability (concentration) and co-localisation - proteins and other molecules need to be located in the same place and the higher the conc. the greater the chance of interaction
- Need to be matching non-covalent interactions
- Competition of alternative partners - will go with the interaction that will form the strongest bonds
- Strength of binding/interaction
How is the structure of a protein specialised to interact with other protein/molecules?
Often have domains within their structure that specialise in specific interaction
What is a domain?
A fragment of a protein that is folded in a particular way and creates a unit that can deliver a specific function
What are some example of a protein-protein interaction domains?
- WD40
- F-box
- SH2
What are some examples of DNA binding domains?
- HTH (helix ten helix)
- HMG - leucine zipper
- Organised to fit into the grooves of the DNA double helix
How can pH affect protein interactions?
- pH can change the charge of the amino acids depending on the pKa
- Can alter the ionic interactions which will destabilise the protein structure and denature it
What are post-translational modifications? How can these affect interactions?
- Changes to proteins after they have been synthesised
- This increases the proteome complexity to provide a diversity in the number of proteins to carry out an increased number of functions
- e.g. proteins undergo phosphorylation, methylation, acetylation etc
- Can disrupt interactions and prevent folding, which changes the structure and can destabilise
What are some modification-specific protein domains?
- Phospho-
- Ubiquitin interacting
- Acetylated lysine interacting
- All of these can only carry out a function or be recognised by a specific molecule after modification
What other group can regulate protein interactions? What is the main example?
- Other ligands
- e.g. calmodulin with Ca2+ which conformationally changes shape to allow the binding of Ca2+, and provide signalling for calcium to carry out other functions
What does DNA polymerase interact with to carry out its function?
- Single-stranded DNA
- Double-stranded DNA
- dNTPs
- Zn and Fe ions - allow catalytic activity
- Regulatory proteins e.g. exonucleases for proofreading and a beta-clamp to allow binding
Summarise protein interactions
- One protein can have many interaction domains
- One protein can interact at the same time with many substrates and ligands
- All of the interactions influence each other and form a complex matrix
How can protein interactions be measured?
- Proteins and other molecules have a reversible affinity
- They are in equilibrium - part always interacts and part does not
- Equilibrium is dynamic and regulated by surroundings
- Identify the proportion/ percentage of protein bound by other molecules on a graph using a dissociation constant
What is the dissociation constant to measure interactions (Kd)?
Affinity is indicated by Ka (dissociation constant) -concentration of ligand required to achieve half (50%) saturation of protein population
Which ligand has the greatest affinity to the protein?
- The pink has the greatest affinity
- Achieves the Kd fastest with a low concentration of ligand added - lowest dissociation constant
- So the pink one has the highest affinity to the ligand and has the strongest interaction
