6BBB0333
L4 – Protein Folding Pathways
What is protein folding and why is it important?
Protein folding: the process of an amino acid chain adopting a three-dimensional structure
Human lysozyme – an example what we might want to think about
- The hydrophobic amino acid residues orientate themselves towards the protein core
whereas the hydrophilic residues project outwards towards the aqueous solution
- Typical folding pattern of globular proteins
- Green - hydrophobic
- Orange - polar
- Red - charged
- All properly dispersed – not patchy (very even)
- If you colour the structure same as sequence - see clusters
- Polar residues on surface, hydrophobic hidden in core.
Membrane proteins – how to adapt to different solvation conditions
- Membrane proteins need to be anchored inside the membrane, but may have other protein
regions sitting on the membrane surface
- The difference in hydrophobicity between membrane and non-membrane must be
accounted for during folding.
,6BBB0333
What is unique about the folding of membrane proteins?
The hydrophobic amino acid residues orientate themselves towards the protein core whereas the
hydrophilic residues project outwards towards the aqueous solution.
- Barrel regions anchoring in membrane. membrane has unique environment - fold in
membrane or before? adaptation to membrane?
What are some questions we need to answer in regards to protein folding?
- Duration of folding, reversibility of folding, requirement of chaperones, do folding pathways
exist, can the intermediates of folding pathways be characterised, the process by which the
hydrophobic and hydrophilic residues end up in the right places, location of folding (during
synthesis? In the membrane?), order of disulfide bonds, membrane protein folding, does the
protein refold inside the membrane? When are chaperones needed? Disulphide bridges –
what order do they fold in and is this important?
What is a native structure determined by? What are the conditions on this?
- The primary structure determines the structure that the protein will adopt, the conditions
include the protein being globular and folding occurring under normal physiological
conditions
When describing the “normal physiological conditions” what does this include?
- Solvent used, pH, ionic strength, presence of ions/ prosthetic groups, temperature etc
What did people know/theorise about folding before the Anfinsen experiment?
- Fischer - long amino acid chains determine the chemical properties of proteins and how they
fold
- Anson and Mirsky (1929-1930) - if slow heating and cooling are used, the protein can be
denatured and then renatured
- Mirsky and Pauling (1936) - first described theory of protein folding and denaturation, held
together using hydrogen bonds
, 6BBB0333
Anfinsen’s thermodynamic hypothesis
What is unique about the Anfinsen experiment?
- Contains 4 disulfide bonds, so 105 possible combos that they could form in and easy to see if
they have or have not formed. Makes folding patterns easy to track throughout the course of
the experiment.
Overview of Anfinsen’s experiment
For globular proteins in their standard physiological environment, the native structure is determined
only the by the amino acid sequence of the protein (ultimate determination of 3D conformation of
polypeptide)
- Used the protein ribonuclease A. For the control experiment, if a reducing agent
(mercaptoethanol) and urea (8M) used to create a random coiled polypeptide chain, then
would refold once oxygen was added and the urea removed, get 100% activity recovered.
But, (test experiment) if you keep the urea and add the oxygen, get irregular disulfide bonds
and once the urea is removed only 1% of the activity is restored.
Urea
- Breaks down non-covalent bonds such as H bonds and ionic bonds that hold together
secondary structure and part of tertiary structure
B-merc
- Breaks disulphide bonds that hold tertiary structure of the protein
- Redox reaction
L4 – Protein Folding Pathways
What is protein folding and why is it important?
Protein folding: the process of an amino acid chain adopting a three-dimensional structure
Human lysozyme – an example what we might want to think about
- The hydrophobic amino acid residues orientate themselves towards the protein core
whereas the hydrophilic residues project outwards towards the aqueous solution
- Typical folding pattern of globular proteins
- Green - hydrophobic
- Orange - polar
- Red - charged
- All properly dispersed – not patchy (very even)
- If you colour the structure same as sequence - see clusters
- Polar residues on surface, hydrophobic hidden in core.
Membrane proteins – how to adapt to different solvation conditions
- Membrane proteins need to be anchored inside the membrane, but may have other protein
regions sitting on the membrane surface
- The difference in hydrophobicity between membrane and non-membrane must be
accounted for during folding.
,6BBB0333
What is unique about the folding of membrane proteins?
The hydrophobic amino acid residues orientate themselves towards the protein core whereas the
hydrophilic residues project outwards towards the aqueous solution.
- Barrel regions anchoring in membrane. membrane has unique environment - fold in
membrane or before? adaptation to membrane?
What are some questions we need to answer in regards to protein folding?
- Duration of folding, reversibility of folding, requirement of chaperones, do folding pathways
exist, can the intermediates of folding pathways be characterised, the process by which the
hydrophobic and hydrophilic residues end up in the right places, location of folding (during
synthesis? In the membrane?), order of disulfide bonds, membrane protein folding, does the
protein refold inside the membrane? When are chaperones needed? Disulphide bridges –
what order do they fold in and is this important?
What is a native structure determined by? What are the conditions on this?
- The primary structure determines the structure that the protein will adopt, the conditions
include the protein being globular and folding occurring under normal physiological
conditions
When describing the “normal physiological conditions” what does this include?
- Solvent used, pH, ionic strength, presence of ions/ prosthetic groups, temperature etc
What did people know/theorise about folding before the Anfinsen experiment?
- Fischer - long amino acid chains determine the chemical properties of proteins and how they
fold
- Anson and Mirsky (1929-1930) - if slow heating and cooling are used, the protein can be
denatured and then renatured
- Mirsky and Pauling (1936) - first described theory of protein folding and denaturation, held
together using hydrogen bonds
, 6BBB0333
Anfinsen’s thermodynamic hypothesis
What is unique about the Anfinsen experiment?
- Contains 4 disulfide bonds, so 105 possible combos that they could form in and easy to see if
they have or have not formed. Makes folding patterns easy to track throughout the course of
the experiment.
Overview of Anfinsen’s experiment
For globular proteins in their standard physiological environment, the native structure is determined
only the by the amino acid sequence of the protein (ultimate determination of 3D conformation of
polypeptide)
- Used the protein ribonuclease A. For the control experiment, if a reducing agent
(mercaptoethanol) and urea (8M) used to create a random coiled polypeptide chain, then
would refold once oxygen was added and the urea removed, get 100% activity recovered.
But, (test experiment) if you keep the urea and add the oxygen, get irregular disulfide bonds
and once the urea is removed only 1% of the activity is restored.
Urea
- Breaks down non-covalent bonds such as H bonds and ionic bonds that hold together
secondary structure and part of tertiary structure
B-merc
- Breaks disulphide bonds that hold tertiary structure of the protein
- Redox reaction
