Chapter 6: Protein Folding, Tertiary Structure, Fibrous Proteins Flashcards
what does it mean to say that a molecule is a ‘dimer’?
it means the molecule or molecular complex consists of two identical molecules linked together.
what is a globular protein?
a compact (relatively spherical) protein that is water soluble and usually involved in stuff like metabolism.
what secondary structures can a globular protein fold into?
alpha helix
beta sheet
what protein/enzyme did Christian Anfinsen study?
Ribonuclease A (RNase A)
Why does Dr Shimko wish Anfinsen had studied a different protein instead of RNase A?
Because the (RNase A) protein that Anfinsen picked is one of the few proteins that has disulfide bonds (and this extra feature makes them harder for students to learn about)
what is a chaotrope?
a small molecule that denatures proteins
what do we use instead of chaotropes?
SDS Page
what are 2 of the common chaotropes?
Urea and Guanidinium chloride
How do chaotropes work?
They steal attention/attraction away from water
water breaks bonds with protein to join the chaotrope; this causes the protein to lose its shape
Do chaotropes work at any old concentration?
No! They must be present at high concentrations.
What was the gist of Anfinsen’s experimental method (in 2 sentences)?
Anfinsen used chaotropes to denature proteins. When he removed the chaotropes, the proteins spontaneously refolded.
What were the 2 major conclusions of Anfinsen’s Denaturation Experiments?
- Proteins spontaneously fold into their native conformation, without facilitation from the cell
- The native conformation of a folded protein is the most thermodynamically favored form
(If the functional state of the protein is the state you arrive at when the protein is at it’s lowest energy, the functional state must be the native state)
what causes proteins to fold?
the hydrophobic effect
what causes proteins to adopt any given shape?
hydrogen bonding
Why did Anfinsen choose RNase A for his protein experiment?
given the tech he had, RNase A was the easiest to work with:
it was easy to get in its natural abundance (from cow pancreas)
it was easy to identify
it was easy to purify into high yields
its activity was easy to detect (experimentally)
What does RNase A do?
it hydrolyzes RNA
it splits up RNA/nucleotide chains into their individual nucleotides by breaking the RNA’s phosphodiester backbone
How did Anfinsen detect the activity of his RNase A?
He’d look to see how thick or thin the RNase A appeared.
RNase A looks thick/viscous when it’s Inactive.
RNase A looks thin when it’s active (the work of breaking up nucleotide chains makes it look thin)
What question was Anfinsen trying to answer when he did his RNase A experiments?
Can proteins fold on their own or do structures/machinery in the cell make them fold?
What were the main 4 steps in Anfinsen’s protein folding experiment?
- purify the protein (outside of the cell)
- denature the protein (with chemicals)
- check the protein’s activity (by thickness)
- try to get the inactive protein to be active again without putting it back in the cell
Why does the RNase A from Anfinsen’s experiment have disulfide bonds?
Because RNase A is a secreted protein that exists outside the cell (in the oxidizing environment) and subsequently has disulfide bonds that other proteins we study do not have.
Is RNase A particularly stable?
Yes. This is part of the reason Anfinsen used it in his experiments
Are Urea and Guanidinium chloride polar chaotropes or NON polar chaotropes?
VERY polar
Why did Anfinsen have to do a control experiment?
To prove that the inactive proteins he renatured really were inactive.
What is a/the hydrophobic collapse?
It’s what happens when a protein gets to be a certain size and its hydrophobic start coming together into what will become the protein’s hydrophobic core.
This process releases caged water
How do proteins fold?
The protein gets made, the hydrophobic effect causes the hydrophobic collapse create the beginning of a hydrophobic core, the molten globule state is adopted, and then the final, most energetically favorable shape is settled upon.
what is a molten globule?
Molten globules are partially folded proteins that have some characteristics of both folded. (or ‘native’) and unfolded proteins.
They represent a transition state between unfolded and fully folded protein
When you see the (y) energy axis of a protein folding funnel, does it represent the energy of the protein or the energy of the system?
BOTH! It represents/INCLUDES the energy of the protein and the system.
What does a protein folding funnel show you?
the thermodynamics of protein folding
Why is the overall energy (of the total system) so high when the protein is completely unfolded?
The caged water around the protein’s exposed hydrophobic regions make the energy high
(more caged water = less entropy)
How do misfolded proteins end up STUCK as misfolded proteins?
The energy needed to unfold and get fixed is higher than the energy of just staying misfolded
do all proteins and conditions present with the same folding funnel pattern?
No. If you change proteins or conditions (i.e. pH), the folding funnel pattern changes.
Folding funnel patterns are characteristic of specific proteins and conditions.
When you see the (x) ENTROPY axis of a protein folding funnel, does it represent the energy of the protein or the energy of the system?
the protein
what’s the avg. energy difference between a denatured protein and a fully folded protein?
about 40 kJ/mole (about the amount of 2 hydrogen bonds
this is why it’s relatively easy to denature them with heat and chaotropes
also why small mistakes/missfoldings can cause such dramatic problems to the organism
on average, are proteins easy to unfold or hard to unfold?
easy
what is T_m represent?
‘melting temperature’
The concentration of chaotrope necessary to get half of a protein to denature
It’s called a melting point even though it actually refers to a concentration
what does it mean to say that a protein sample has ‘two state unfolding’?
it means that the transition folded and unfolded is abrupt; a sample will unfold slowly, then all of a sudden, the remainder of the sample will fold, too
On a graph this will appear as a very steep slope
what does cooperative protein folding/unfolding mean?
Protein peer pressure.
In a given structure, once part of a protein unfolds, it will influence the rest of the structure to unfold.
Similarly, once an entire protein unfolds, it will encourage other proteins to unfold
Is T_m constant or is it a property of each given protein?
It’s property of each given protein
Does a high T_m mean that a protein is MORE stable or LESS stable?
More
High T_m means a higher concentration of chaotrope is needed to denature the protein.
how does the hydrophobic effect impact protein folding?
it triggers unfolded proteins to fold such that the hydrophobic regions are AWAY from the aq environment, the hydrophilic regions are EXPOSED to the aq environment, and the amount of caged water present is greatly reduced.
what is a fibrous protein?
a protein composed of repeating secondary structures packed together
are fibrous proteins soluble?
no. not really.
what is the general function of fibrous proteins in living things?
its purpose is primarily a structural
is keratin a helix or a beta sheet?
alpha helix
where in the living thing is keratin usually found?
hair, nails, feathers
what is the general function of silk in living things?
spider webs / fibroin
is silk a helix or a beta sheet?
beta sheet
where in the living thing is collagen usually found?
skin, bone matrix, tendons, blood vesicles
is collagen a helix or a beta sheet?
neither. Collagen adopts a TRIPLE helix structure
why is keratin referred to as a dimer?
because it has a coiled-coil structure (where 2 identical molecules are linked together)
what is special about keratin’s alpha helix (with regard to the bonds in the tertiary structure)?
It has a lot of disulfide bonds
they occur between the protein’s coiled coil and stablizes the structure
what is special/exceptional about keratin’s coiled-coil structure?
unlike other proteins with coiled-coil structure, it is almost entirely hydroPHOBIC
are all parts of the keratin structure equally hydrophobic?
no. the most hydrophobic regions aggregate in the protein’s core and the slightly less hydrophobic regions aggregate near the protein’s surface.
what is the role of charges in the keratin 2o structure?
The orientation of charges facilitates the keratin’s packed shape
why is it that keratin can have disulfide bonds?
keratin is extracellular/exists in an oxidizing environment
what (structure) do packed keratins give rise to?
toilet paper rolls full of goo.
intermediate filaments (exp. hair strands)
how are the beta sheets in silk arranged?
they are stacked on top of one another and each sheet is bonded to the next sheet by H bonds
what comprises the repeating amino acid pattern for silk?
glycine and alanine
(with regard to amino acid size) what makes silk’s pleated sheet different from the other pleated sheets we’ve learned about?
silk’s pleated sheets have tiny residues (alanine and glycine). pleated sheets usually have large/bulky residues and/or residues with beta branches.
what forces hold the SIIIIIIIIIIIILK pleated sheet layers together?
the layers are packed/bound together via the hydrophobic attractions between the alanines and glycines
like a lasagna
are silk’s beta sheets parallel or antiparallel?
antiparallel
can silk backbones be rotated/stretched?
only a little. The strands are already essentially in extended conformation.
is the collagen triple helix made of alpha helices?
.NO!!!!!
it’s made of special left handed helices that are wrapped around each other in a right handed fashion (like a rope),
where (in living things) is collagen usually found?
bone, skin, tendons, blood vesicles, cartilage
why does collagen have/need post translational modifications?
it needs modified lysine (allysine) to enhance structural stability
it needs modified proline (hydroxyproline) to facilitate the left handed helices in the triple helix
what is the consensus sequence for collagen?
Gly-X-Y:
glycine-proline-hydroxyproline
what makes the collagen helix RESIDUES different from the helix residues we learned about earlier?
alpha helices (like the ones we originally learned about) don’t ever have proline or glycine.
collagen has proline AND glycine. and it has them repeating over and over again.
why does the collagen helix need hydroxyproline?
the triple helix shape needs both the ring flips from proline AND the ring flips from hydroxyproline in order to maintain its structure and stability
what (structure) do packed collagens give rise to?
a collagen fibril
what does the term ‘counter woven’ describe?
collagen’s rope like left handed helices wrapped around each other in a right handed fashion
what does ‘cross linking’ in collagen involve?
a bond between multiple layers of collagen chains that is facilitated by attractions between modified amino acids (like allysine)
what is allysine’s role in collagen’s cross linking?
it is one of the modified amino acids in the amino acid chain that binds (cross links) one collagen molecule to another
It loses and amine group, becomes and aldehyde, gets nucleophilic attacks and thus becomes the crosslink bond to the nucleophile (which is a Schiff base from the other later of collagen)
why do you need vitamin C to make collagen (and avoid scurvy)?
blood vesicles need collagen, collagen needs vitamin c to perpetuate the chemical process for making hydroxyproline
collagen specifically needs to be able to convert Fe2+ byproduct (from hydroxyproline making) back into Fe3+ (so it can keep hydroxyproline making)
