Lecture 2: Folding and Flexibility Flashcards
What are common features of proteins?
- Outside tends to be exposed to hydrophilic
- Inside are rather hydrophobic (not completaly)
- Often globular form (BUT many many exeptions)
NOTE: hydrophobic packing = driving force for protein folding
What kind of protein structures make use of hydrophobic interactions?
1) Globular proteins
- hydrophillic (polar, charged) amino acids outside, exposed to water
- hydrophobic amino acids packed together in the middle
2) Beta-alpha-beta motif
- hydrophillic aa at top of the helix
- hydrophobic aa packed against hydrophobic aa of the beta sheets (shielded from water)
What exactly is the hydrophobic effect?
- Water tends to build hydrogen network -> each hydrogen bond releases energy
- If we wanted to break, due to an apolar molecules, the bonds it would require energy = energetically unfavourable
- In order to minimize the disruptive effect we need to reduce the surface of interaction
What is meant by active protein conformation - what, name + what is the opposite and what causes it?
- Active conformation of proteins = native state, how the proteins occur in nature
- Unfolded, denatured state:
- high temperature
- high pressure
- high concentration of certain chemicals
=> disrupts the structure and neighboring water network
What do we see in the diagram?
= graph depicting multidimensional free energy surface
- it shows what is the energy freed during different states = y axis
- Different folding states = x and z axis
Folding difference = different conformational changes
- lower energy and more H-bonds = prefered, good packing
- tight packing = costs entropy for the protein (water gets more entropy)
=> Folding state is an equilibrium on DeltaG level between folding & unfolding
Elaborate on what you see in the picture:
Protein will start at the top as unfolded -> goes down into intermediate states (hydrophobic interactions) -> native state, functional
- If something happens to the system (e.g. getting into new chemical environment, interacts with agents) => may not return to native state but instead gets into a different state
- e.g. amorphous aggregates - packed tightly together, disfunctional, amyloid fibrils - could be advantagous (e.g. in cytoskeleton) or dangerous (e.g. in membranes, cytosol)
NOTE: absolute minimum = native states
What do we mean by “Molten Globule”? What more can be said?
= first step of so called Hydrophobic collapse, intermediate between unfolded and native state (folding intermediate in a local minimum on free energy landscape)
- globular structure - what we get when polypeptide gets out of ribosomes
- Starts building the secondary structures - BUT more loose with less interactions than native
- Extremely fast (experiments usually do NOT pick it up)
- Hydrophobic collapse inward - energetically more favourable than being unfolded
What do we mean by “native state”?
= a local minimum on the free energy landscape
- Picture:
- green = native states (proteins tend to have different, still preferred, conformation), red = all other states (intermmediate, missfolded, aggregates)
- there are many steps that could take us to the same point
What are the characteristics of global minimum?
= deadly packed, non-functional
- only one, hard to get out of
We hypothesized Levinthal’s Paradoxon - what is that?
We may assume that amino acid can be in either of 3 states (neta sheets, right and left alpha helices) -> we may calculate the amount of conformation possible (however, some could be non-functional ofc.)
- Unfolding and denaturing = extremely fast
=> Proteins fold within seconds
- Hard to predict the 3D structure from primary sequences - there are too many options
- we have to understand the underlying mechanism of folding
What does this picture demonstrate?
Number of found (relevant) unfolded structures is much smaller than the number of possible unfolded structures
- If the number of relevant (found) unfolded structures increases proportionally with folding time, only 10(9) protein structures need to be simulated instead of 10(47)
=> There must be some guidance in the unstable, less-funtional folding mechanisms (we need to limit the number of structures that would be possible in netherlands
There are Folding Helper Proteins - what are the 3 major obstacles that they help with?
1) Forming of incorrect disulfide bonds
- Can be formed and broken down to constrain and release
- we have specific enzymes for both
2) Isomerization of proline residues
- Isomerases
3) Aggregation of intermediate due to exposed hydrophobic patches
- Chaperones - large complexes that help out with correct conformation
Why do we need the disulfide bonds to keep building and releasing?
In the picture we have a polypeptide with 6 cysteines -> goes through multiple intermediate steps as the polypeptide chain grows
- notice the first disulfide bond stays while the other keep changing depending on the folding stage (until reaching native state)
- This ensures the proper build up of all the secondary structures
- Protein Disulfide Isomerase (PDI) enzymes help out
What are the 2 forms of Proline?
Most peptide bonds are “trans” -> BUT proline is able to form the “cis” conformation more often than others
=> side effects on the backbone and side chains e.g. cys proline can build tighter bonds/loops
- Cyclophilin = protein that allows this isomerization
What is the issue with Molten globule?
Being in the state of molten globule make it difficult to transition into the native state - a lot of energy is required
=> We need the Folding Helper-proteins
What kind of Folding Helper proteins can help with transition from molten globule to the native state?
Chaperones
- throughout the folding aggregation may occur due to hydrophobic residues
- These proteins can provide protection
- especially helpful in high temperatures (found to be upregulated in bacteria in such conditions)
- Heat-shock proteins: Hsp70, Hsp60, Hsp10
- 7-meric ring (7 subunits) - we have 2
- intermediate domain uses ATP hydrolysis
Look at the structure of the GroEL/GroES complex:
What is the GroEL/GroES mechanism?
- Unfolded protein binds to the donut without the lid
2) Release of 7 ADP and GroES on the other donut
3) GroES on side 1 closes the lid and 7 ATP bind to side 1
4) Hydrolysis: ATP -> ADP + Pi
5) 7 ATP bind to side 2
6) Hydrolysis: ATP -> ADP + Pi
=> GroES opens
=> folded protein leaves
=> ADP exits
=> new protein binds- We’re at 2 again
What exactly does GroEL/GroES complex do?
- GroEL/GroES they don’t know every protein -> correct folding depends on the amount of unfolding that occurs
- usually misfolded proteins should unfold -> get a second chance on folding => GroEL/GroES increase the unfolding (need to apply energy to break it)
- ATP is aided by the passive unfolding that occurs anyway
Seems like it helps with folding BUT ACTUALLY induces forced unfolding
Sometimes we can have multiple native states - could you think of a type of protein with alternative foldings?
Prion = comes from PRotein and infectION
- function is unknown, found in the brain
- Has two conformation:
- normal alpha-structure
- Harming beta-structure => can attach to healthy proteins andcan refold them
- NOTE: look at recommendations on hw to get rif of it
What can prions cause in the brain?
- Beta structures can aggregate and form plaques => cells die and sponge like structures are left behind
Creutzfelds-Jacob Disease (CJD) = rapid dementia, muscle stiffness, ataxia, hallucinations
Kuru = tremors, loss of coordination, emotional instability
- occured in Papua New Guinea -> tribes that ate the brain of the dead
Bowine Spongiform encephalopathy
- eating meat that has the proteins (they are quite tough)
What is one misconception about native state of a protein?
Even after reaching the native state -> proteins remain active, flexible
- NOT shown in our measures since they can only capture stable images
- we need the movement - especially true for loop regions (as opposed to beta sheets which are quite stable)
What are B-factors?
B-factors = calculated factors/numbers of how much an atom moves around the depicted position (x,y,z)
- each atom has this number -> vary a lot
- high value = more movement
- We can see that within secondary structure elements the B-factor goes down - less flexible
How is flexibility of a protein connected to its function? You may illustrate it on a specific example.
Nature uses it for conformation changes =>
Estrogen receptor - member of nuclear receptor family
- involved in: binding of DNA (Transcription factor), binding of natural ligands - estradiol E2
- Has active and inactive state (there are agonists and antagonists that put them into one of these states)
