Tertiary structure and protein stability Flashcards
The tertiary structure is the overall pattern of…
Tertiary structure is the overall pattern of folding of the whole polypeptide chain
- The simplest possible tertiary structure is continuous secondary structure
- ⍺-keratin is totally ⍺-helix
- Fibroin (a β-keratin) is totally anti-parallel β-sheet
- Collagen (tendon, bone and connective tissue) has a unique triple-helix structure
- Collagen structure requires sequence with repeating units of three amino acids. -Gly-Pro-X-
- Secondary structure is rigid, so these are fibrous proteins
Most proteins are globular, what does this require the polypeptide to do?
Most proteins are globular: this requires the polypeptide to fold back on itself
- Folding requires “breaks” in secondary structure, which is rigid
- Clusters of 2-3 secondary structure breakers (Gly, Pro, Asn, Asp or Ser; GPNDS) in a run of 4 AAs
- Allows for flexible loops and turns where polypeptide can change direction to allow folding
What is the hydrophobic effect?
The hydrophobic effect is a major force driving protein folding
- Folding the protein encloses most of the non-polar amino acids in the core
- Amino acids are drawn with space-filling atoms, colour- coded polar or non-polar
- Non-polar AAs (red) group together to minimize contact with H2O (hyrdophobic effect)
- Polar AAs (green) form the outer layer, interact well with surrounding H2O (good H-bonding) or with ions in solution
Amino acids pack together with jig-saw puzzle fit, why is this important?
- The side chains interlock to maximize the number of close atom-to-atom contacts
- Close contacts attract by weak Van der Waals forces
- Aka london dispersion forces
- 0.1 to 1 kJ/mol per contact
- Good fit makes hundreds of close contacts in macromolecule, and helps to hold structure together
- Poor fit only makes a few contacts
What is the way a protein folds dictated by?
Summary
the way a protein folds is dictated by the sequence of its amino acids
- Amino acids “elect” secondary structures
- “Breaker” AAs allow for folding, introduce flexible sections
- Distribution of non-polar amino acids in sequence determines which parts fold inwards
- Polar amino acids interact well with aqueous surroundings
- Pattern of large and small side chains is arranged so that secondary structure components (e.g. helices) pair up with best possible fit
A “balancing act”; many different forces in play, protein reaches the stable native state
Proteins fold into a limited number of tertiary structure patterns. What are they?
- proteins consisting of mostly ⍺-helical segments
- proteins consisting of mostly β-strand segments
- proteins with alternating ⍺-helical and β-strand segments
What will a sequence with mostly groups of ⍺-helix-forming amino acids form into?
A sequence with mostly groups of ⍺-helix-forming AAs will fold into an ⍺-helix bundle
- Small clusters of breakers set the limits of each helix
- Non-polar AAs every 3 or 4 places in the helix make a non-polar patch or stripe, e.g. - PPNPPNNP-, which fold to inside of bundle
- AAs that prefer β-sheet are present, but scattered
- Myoglobin is a bundle of 8 ⍺-helix sections
- Bundles of 6-8 helices appear more complex because the helices splay apart
What will β-sheet-forming amino acids in majority fold into?
β-sheet-forming amino acids in majority fold into antiparallel β-sheet
- Anti-parallel sheet is more stable because H-bonds are arranged in straight line
- Side chains project out of the sheet; odd on one side, even on other side
- Sheet can be non-polar on one side, polar on the other side
A β-sheet that is polar on one side and non-polar on the other wraps around to enclose which face inside?
A β-sheet that is polar on one side and non- polar on the other wraps around to enclose the non-polar face inside
- A small sheet (3-5 strands) makes an open field
- A larger sheet (6-8 strands) wraps all the way around to form an antiparallel β-barrel
- This example is green fluorescent protein
What can sequences which alternate structure β-⍺-β-⍺ form?
sequences which alternate structure β-⍺-β-⍺ can form parallel β-sheet
- Helical sections connect the strands, which all run in the same direction
- Helix lies above or below the plane of the sheet
- Parallel β-sheets are less stable (angled H-bonds), so must be sequestered away from H2O
- Usually buried in centre of protein, thus made up of mostly non-polar amino acids
If all the helices lie on one side of the sheet what does it form?
If all the helices lie on one side of the sheet, the sheet wraps around to form a parallel ⍺β-barrel
- The -sheet forms the central barrel, surrounded by the connecting ⍺-helices
- Example is the enzyme triose phosphate isomerase.
Helices on both sides of the sheet form what?
Helices on both sides of the sheet give the parallel ⍺β-sandwich structure
- Sandwich filling is the non- polar β-sheet between two layers of ⍺-helix
- The β-sheet is often twisted for better packing
- Example is part of the enzyme lactate dehydrogenase
Many larger proteins fold up in different sections called…
Many larger proteins fold up in different sections called domains
- Each of the structures outlined above forms from a chain of 10-20 kDa
- Larger proteins fold up in 10-20 kDa sections; each section is called a domain
- A protein of 50 kDa may have 3 or 4 domains
- Each domain may adopt a different folding pattern
- Thus, larger proteins are often modular in nature
- Example is lactate dehydrogenase with two ⍺β-sandwich domains
What is protein stability and function?
- The normal folded state of a protein is called its native state, essential for proper function
- When unfolded, a protein is said to be denatured, loses all function
- Covalent bonding links amino acids in a chain in a specific sequence
- Non-covalent interactions dictate folding pattern and stability
- Hydrophobic effect and van der Waals effect are the most important non-covalent interactions
- Hydrophobic effect locates non-polar amino acids in core of folded protein, avoiding unfavourable interaction with H2O
- contributes ~50% of total energy stabilizing native state
- –5 kJ/mol per CH, CH2 or CH3 moved out of contact with H2O
- Polar amino acids can face exterior, where interact well with H2O
What is van der Waals interaction?
- van der Waals interaction is a weak electrostatic attraction between atoms that are close, but not covalently bonded to each other – aka London dispersion forces
- Random fluctuations in distribution of nucleus and electrons create short-lived dipoles, which induce dipoles in close neighbours
- Free energy of interaction:
- Atoms too close together [A] repel strongly (positive free energy)
- Atoms at ideal distance [B], close to each other
- Atoms further away are attracted [C]
- Force fades away when atoms are more that 2-3 diameters apart [D]
- Weak interaction is only effective if many atoms are in close contact
What can polar interactions help stabilize?
Polar interactions may also help stabilize the correct folding of a protein
- H-bonds may form between donors and acceptors that line up in the folded protein
- Ion pairs: strong electrostatic interaction of negative side chains which pair up with positive side chains that are nearby in the folded protein - salt bridge
- Any polar interactions important for maintaining structure are usually in protein interior, rather than on surface
- most polar groups face the exterior:
- charged AAs pair with ions in external solution
- H-bonding AAs bond to H2O in surroundings
What do disulfide bonds help?
Disulfide bonds help to hold together the tertiary structure of some proteins
Disulfide bonds form when pairs of Cys –SH groups react with O2, releasing H2O
- This makes a strong covalent bond to help hold the folded protein together
- Few proteins have disulfide bonds; mostly proteins designed to function outside cells since O2 needed
The primary structure contains all the information required for…
The primary structure contains all the information required for the secondary and tertiary structure of a protein
- Ribonuclease is an enzyme which catalyses hydrolysis of RNA
- 8 Cys make 4 specific disulfide pairs in the properly folded protein
- Ribonuclease is denatured (catalytic activity is lost) by placing it in 8 M urea solution with 2-mercaptoethanol
- Urea weakens hydrophobic effect, so allows protein to unfold
- 2-mercaptoethanol is a reducing agent that converts disulfides back to the original unlinked Cys-SH groups
How can ribonuclease refold to its original structure?
Ribonuclease can refold to its original structure when urea is removed
- First remove urea so ribonuclease refolds
- Then expose to O2 so disulfide bonds form
- Refolded enzyme has normal catalytic activity
- Refolded enzyme has the original pairs of Cys in disulfide bonds
- If exposed to O2 before refolding, disulfides pair up randomly –wrongly folded, inactive
