Protein Folding & Structure Flashcards
What are the brief steps involved in Anfinsen’s experiment?
What were the results?
What was the conclusion made?
What is the basis of protein dynamics?
What does protein dynamics allow for proteins?
Add urea (H bonds) and denaturant (disulfide bonds) to RNAse A & remove
Reformed its original native & catalytic structure
Primary structure determines protein structure
Proteins constantly in equilibria within F and U states - even within those states (intermediates)
Fast turnover (Kcat), flexibility, control protein availability
How do proteins fold?
What is the basis of this?
Why is folding unlikely to be a random search?
What do intermediates do in the folding pathway?
What are the 3 ideas of protein folding models?
What do they all have in common?
Hydrophobic Effect
Hydrophobic residues form a core that stabilises the folded state - hydrophilic residues on the exterior that form hydrogen bonds with water
Amount of amino acid sequences possible & amount of time taken to try these combinations
Assist folding process - lower energy states (free energy) so more favourable towards folded
- Formation of secondary structure elements
- Hydrophobic collapse
- Nucleation-condensation
Formation of some protein core (secondary structure or hydrophobic res) for the rest of the protein to assemble around
What is the folding funnel driven by?
Describe the unfolded state
Molten globule state
Folded/native state
Why is the molten globule state dangerous?
How can this be prevented?
How can proteins be viewed?
Protein dynamics between U & F states/hydrophobic effect
High entropy & high energy state
Local minima/low energy trophs - frustrated state of well formed secondary structures but no tertiary
Low energy & low entropy state
Energy is required to leave the state - as a result more favourable for protein to aggregate, oligomerise & form amyloid fibres
Chaperones
Partly stable - balancing between folded & unfolded states
What are class I chaperones?
Class II?
What is the structure of GroEL/GroES?
What is the structure of amyloid fibril deposits?
How do amyloid fibrils cause Huntington’s?
Alzheimer’s?
HSP70 type
CHaperonins - GroEL/GroES
2 x 7 membered ring - barrel structure with cap
Heavy beta sheet network
Intranuclear inclusions in neurones are polyubiquinated
Alpha-beta plaques deposited in neurones
What kind of protein is the alpha 1 antitrypsin?
Why is it metastable/how does it become fully stable?
How can this cause disease on mutation?
Protease inhibitor
Insert reactive loop into target protease - loop becomes part of 6-stranded beta sheet
Insert into molecules that aren’t its toxic substrate - end up forming polymers of subtrates - liver & lung disease
What is the rule of thumb for peptide bonds?
What are 2 characteristics of a peptide bond?
Why are most peptide bonds with proline included not trans?
What is the phi bond between?
Psi?
How is the peptide bond conformation defined?
Why are staggered peptide bond orientations more favourable?
CORN - CO - Ca(R)(H) - N = L-form
Planar
Partial double bond character
Causes steric interactions between side chains
Ca-N
Ca-CO
Degree of freedom/rotations are restricted to phi & psi movements
Minimise interactions
In an alpha helix, where does the hydrogen bond form?
What does peptide bond formation/secondary structure depend on?
How do the side chains project?
Why?
What are the characteristics of the 3-10 helix?
Pi helix?
What is the net dipole moment?
i of O (COOH) & i+4 of N (NH2)
Primary sequence
Outwards
Minimises interactions with side chains on the same helix - energetically more favourable
Overwound - positively supercoiled/longer
Underwound - negatively supercoiled/shorter
N(+) -> C(-)
How are the hydrogen bonds oriented in an anti-parallel beta sheet?
In what directions are the side chains?
What are anti-parallel beta sheets connected by?
What are the directions of the side chains in the parallel beta sheet?
What is a mixed beta sheet?
What are its 2 characteristics?
What amino acids are the most favourable for a beta turn?
Why?
Antiparallel
Alternative above & below
Short loops (hairpins)
Short chains on neighbouring strands point in the same direction
Mixture of both antiparallel & parallel beta sheets
Not flat, each strand has right handed twist
2nd Pro and 3rd Gly
Don’t form a hydrogen bond but make abrupt turn
What is a serine protease inhibitor (serpin) made up of?
In what conditions are disulphide bonds made?
What are the weaker forces?
Strongest?
What other force is involved in protein structure?
What is a Zinc finger?
What is a greek key?
Mixed alpha-beta fold: 3 beta, 9 alpha & reactive centre loop
Oxidising (oxidising agent) - coupled reduction oxygen
Ionic/electrostatic, then H bonds
Disulfide (covalent)
Hydrophobic - arising from non-polar residues in core stabilising protein structure
Zinc ion (large & soft) binds to 3 histidines & 1 water
2 hairpins (antiparallel beta sheets)
What are the steps of primary structure -> quarternary?
What are the 4 steps to GroEL/ES?
Why is a hydrophilic environment favoured?
What are the 3 subunit domains of GroEL/ES?
Primary, secondary, motifs, domains, tertiary, q
- Partially folded/misfolded protein enters GroEL cage & binds to hydrophobic surface near cap
- ATP & GroES binds - forms lid
- GroEL doubles volume on GroES binding & becomes hydrophilic environment
- ATP hydrolyses, GroES released & protein released
Favours protein binding
Apical = for protein & GroES binding
Intermediate = hinge connecting 2 domains
Equatorial = mediates ATP binding
What is an intrinsically disordered protein?
What is their structure defined by?
Why can they form very limited hydrophobic cores?
How do they gain structure?
What do IDPs show about proteins generally?
How does A-synuclein IDP cause disease?
Proteins that don’t have defined 2D/3D structure - regions of disorder & linker sequences
Interacting partner
Few hydrophobic residues - lots of hydrophilic
Binding to a target - mediates protein-protein interactions
Interactions are mediated by structural plasticity (increased functional space due to disorder)
Point mutations form amyloid fibrils into lewy bodies - bind to lipid membranes
What does the hydrophobic effect rely on?
How does the hydrophobic effect play part in IDPs stability?
What problems did Linus Pauling recognise in the 40s?
What did Levinthal in 1969 realise about protein folding?
What was the control in Anfinsen’s experiment?
What did this show?
Solvent, salt conc, pH, temp
Compensates for loss of entropy when binds
Huge amount of conformational space but only 1 solution to a native protein - assumed very rapid & spontaneous event
Random conformational search isn’t the answer - many combinations which can be narrowed down by steric hinderances & Ramachandron plot
- folding happens in 1 step in microsecond
Oxidised unfolded protein 1st then renatured 2nd (experiment was other way around)
Disulphide bonds formed first before denaturant removed - bonds formed incorrectly & so protein was properly unfolded at denaturation
What is the diffusion collision folding theory for alpha helices & folding?
Why is this less suited towards beta sheets?
How can NMR be used to observe protein folding?
How can CD be used to observe protein folding?
What was observed in speed of folding between a helices & beta sheets?
4-helix bundle where helix-helix collision resulting in collapse into native structure
Beta sheets need hydrogen bonds between adjacent sheets to stabilise
Monitor hydrogen-deuterium exchange of NH amide backbone protons
A-helix & beta sheets are chiral so can measure change in absorption as a function of denaturant, conc or temp
Alpha helices fold faster - don’t require another element to form hydrogen bonds - forms internally
How does the optical tweezer method observe protein folding?
What was observed from this method?
What was the conclusion?
What are the 4 structural characteristics of molten globules?
What is the idea of molten globules being a trapped state?
What is the conclusion about molten globules?
Binding both sides of protein to a bead & stretch to unfold - measure force required
2 forces: abrupt shift & smaller shift
Stable core of protein folds first (abrupt) & then rest folds around
Secondary structure, no tertiary structure, compact & loose hydrophobic core
Require sufficient energy from troph/local minima to move back into intermediate state in the pathway: U -> I (-> T<-) -> F
Partially stable conformation (intermediate) but trapped states cannot be avoided when going from intermediate -> folded state
Why can MD not simulate protein folding?
What is MD used for?
What are the problems for simulating protein folding?
Doesn’t account for the hydrophobic effect
Protein energetics
Accuracy of the force field, large conformational space, local minima, parameters from small molecules/proteins
