Which statement is true for the
native state of a protein?
A. It is a single rigid structure.
B. The low entropy of the native state makes it
energetically favourable.
C. It will maximize the number of hydrogen
bonds that can be formed.
D. It may contain knots in the polypeptide
chain
A. It is a single rigid structure.
❌ False.
Proteins in their native state are dynamic, not rigid. They undergo small conformational fluctuations that are important for function.
B. The low entropy of the native state makes it energetically favourable.
❌ False.
The native state does have lower entropy (more order), which is actually unfavourable. Folding is driven mainly by things like the hydrophobic effect and favorable interactions, not by the entropy of the protein chain itself.
C. It will maximize the number of hydrogen bonds that can be formed.
❌ False.
Proteins do form hydrogen bonds, but the native structure does not simply maximize them. Many potential hydrogen bonds instead form with water. The structure reflects an overall free-energy minimum, not just maximum H-bonds.
D. It may contain knots in the polypeptide chain.
✔ True.
Some proteins are known to fold into structures where the polypeptide chain forms a knot, though this is relatively rare.
✅ Correct answer: D.
Are forces in protein weak or strong?
weak, non covalent
Native state can be destroyed or denatured by what?
heat
pH
detergents
organic solvents/molecules (Acetone, Guanidine HCl)
Draw the structure of two denaturants: guanidinium ion and urea
what are their properties?
chaotropic agents (reduce hydrophobic effect)
water soluble (can form H bonds with protein backbone)
disrupt hydrophobic interactions
What are two reducing agents and their properties?
beta mercaptoethanol (BME)
dithiothreitol (DTT)
reduce disulfide bonds
become oxidized as part of the reaction (disulfide exchange, cystine to cysteine, -S-S to SH + SH (2 BME or DTT donates their hydrogen, then become one thing)
Denaturation is a cooperative process, meaning what?
Describe this process and draw the graph of denaturation by temperature, and denaturation by urea and guanidine Hcl)
Cooperative means that parts of the protein influence each other during unfolding
transition from folded to unfolded occurs over small range (and can be tracked by spectroscopic markers)
midpoint of transition (Tm) is characteristic of both the denaturant and the protein
unfolding part of the protein decreases the energy required to unfold the rest of the structure
“…the native conformation is
determined by the totality of
interatomic interactions and hence
by the amino acid sequence,
in a given environment”
Describe the process of renaturation of denatured ribonuclease (Christian Anfinsen)
removal of denaturants may allow protein to refold (if no covalent bonds have been affected)
if disulfide bonds reform before denaturant removed, protein may be locked in wrong conformation
note: urea denatures, BME cleaves disulfide bonds, both cause denaturation
removal of denaturant (urea) AND reductant (BME) allows protein to renature and reform disulfide bonds IN PRESENCE OF OXYGEN (native)
BUT
removal of BME allows disulfide to reform in denatured protein
then the removal of urea generals enzymatically inactive protein in which disulfide bonds have formed at random
adding small BME to scrambled protein in absence of O2 catalyzes conversion to active enzyme through disulfide interchanging reactions (allow native disulfide bonds to form
native
What is the formula for total combinations of disulfide bridges for even numbers of Cys, where n is the number of Cysteine residues?
(2^(-n/2) n!) / (n/2)!
Protein folding is a what kind of process
cooperative
energic collapse to folded state
What is the folding pathway process
form secondary structures, motifs, domains
just before final tertiary structure, molten globule state (hydrophobic core, secondary structures present, tertiary state not set, dynamic)
Free energy of folding can be visualized how
free energy funnel
energy y axis, entropy x axis
unfolded states (top) have large conformational entropy and high energy, but as folding progresses, number of states present decreases and conformational entropy decreases
some structures may be relatively stable but do not represent native structure
native, folded state (bottom) has lowest conformational entropy
Misfolding can produce stable structures which are not native.
This may be associated with disease states:
amyloidosis (Huntington’s, Alzheimers)
Prion diseases (BSE, scrapie)
Collagen defects (Osteogenesis imperfecta)
Protein folding processes may be assisted by other proteins
Give examples of molecular chaperones and isomerases
molecular chaperones
- heat shock proteins (Hsp70/40; Hsp90)
- chaperonins
isomerases
- protein disulfide isomerase (PDI)
- peptide prolyl isomerase (PPI)
What are characteristics of chaperones/chaperonins?
misfolded proteins expose hydrophobic regions which can aggregate
chaperons isolate misfolded proteins so they can’t interact and give them a chance to refold
E is typically req (APT hydrol) to unfold protein substrates, so they can partially unfold
process is cyclic until proper folding occurs
process is kinetic, not thermodynamic (native structure is still determined by protein’s intrinsic thermodynamics)
Describe the Hsp70 chaperone system, which uses ATP to unfold and refold
1) ATP binds DNKAK (Hsp70), DNAJ (Hsp40) brings misfolded protein to DNAK, DNAK recognizes exposed hydrophobic region
2) ATP hydrolysis, stimulated by DNAJ tightens binding, allowing partial unfolding of protein
3) GrpE promotes nucleotide exchange, removing ADP from DNAK, and new ATP binds to DNAK, causing DNAK to release substrate, then the protein will attempt to refold properly
if it misfolds, cycle repeats
How big is the GroEL/GroES complex?
What size proteins can fit inside?
184 A tall
140A diameter
small central pore is 10 A
C7 symmetry
proteins up to 60kDa can fit in the internal cavity
Protein Disulfide Isomerase will break disulfide bonds in protein substrates. What amino acids are likely found in the active site?
C
Describe the GroEL/GroES complex chaperone process
1) misfolded protein binds GroEL ring, then 7 ATP binds that ring, ring is now in cis ring, oppsoite ring in trans
2) GroES cap binds to GroEL, conformation changes and substrate is released into the internal hydrophilic cavity which prevents aggregation and gives it a chance to fold
3) in cis cavity, protein has 10 seconds to fold, then 7 ATP mole are hydrolyzed which weakens GroEL-GroES interaction
4) a ew substrate binds the trans ring, 7 more ATP binds there to set up the next cycle. ATP binding causes GroES, ADP, and substrate to release from the cis ring (usually its better folded)
now, the trans ring turns cis and vice versa
What does protein disulfide isomerase (PDI) do?
catalyzes the shuffling of disulfide bonds to form the correct bonds of the native conformation
reduced PDI makes native S-S bonds (isomerase property)
oxidoreductase property: oxidized PDI and reduces protein creates the oxidized (native) protein (and reduced PDI)
What does peptide prolyl cis-trans isomerase (PPI) do?
proline residues may adopt a cis or trans peptide bond
10% of proline residues have cis peptide bonds
adopting a cis bond is NOT spontaneous, and PPI needs to assist in that reaction
Which statement is false regarding chaperones?
A. They usually consume ATP as part of their action.
B. They change the native state of the protein substrates.
C. They act on many different proteins.
D. They prevent aggregation of unfolded proteins.
B
