Proteins - T&Q, Forces and Folding Flashcards
(31 cards)
What is the tertiary structure of proteins?
Describes the folding/organisation of secondary structure elements
Can be a/b or both and is very unique
How can we determine the positions of atoms in proteins?
X-Ray crystallography - A crystal of the molecule is imaged resulting in a diffraction pattern (atomic resolution not achieved)
NMR spectroscopy - interactomic distance measurements and geometric constrainsts are used to build a 3D image
Cryo-electron microscopy - sample is cooled (-196 C) so fast it assumes a vitreous (glasslike) state = retains native state for viewing
Where are amino acids normally found in globular proteins?
Non-polar residues - interior of the protein
Uncharged polar residues - on the surface but also occur in the interior
Charged polar residues - on the surface
Due to the hydrophobic effect
What are groupings of secondary structure elements called?
Supersecondary structures or motifs (special folding)
Name some supersecondary structures/motifs?
- Beta-alpha-Beta (the helix connects two parallel beta strands)
e. g. triose phosphate isomerase - Beta hairpin (antiparallel strands connected by tight turns
e. g. erubotoxin - Alpha-alpha (two successive antiparallel helices packed against each other with their axes inclines
e. g. calmodulin - Greek-key motif (a beta hairpin folded over to form a 4-stranded anti-parallel beta sheet)
How are most proteins classified?
All alpha helices
All beta sheets
But most contain a mixture:
A/B mixed together
A/B with segregated regions
How are the a, b and a/b classes further categorised?
Via topology - according to how their secondary structure elements are connected:
Beta barrels x3
2 are all B with beta hairpin motifs
1 is an a/b barrel with overlapping BaB motifs
What do larger polypeptides form?
Domains
Each domain consists of 40-200 amino acids with an average diameter of 25 Å
e.g. Glyceraldehyde 3-phosphate dehydrogenase
Some domains are structurally independent - can be separated and be stable
Other multi-domain proteins have binding sites occupying clefts between domains
Rossmann folds often act as nucleotide binding sites (identification of this fold = clue to the function of the protein)
Describe quaternary structure?
Many polypeptide chains non-colvalently assembled
Larger than 100 kDa
Describe subunits within quaternary structures?
The subunits associate noncovalently
Proteins with more than one subunit = oligomers and their identical units = protomers
Homo oligomer - one type of protein subunit
Hetero oligomer - different types of protein subunit
How are subunits within quaternary structures arranged?
Symmetrically
Each protomer occupies a geometrically equivalent position in the oligomer
Due to only L residues = rotational symmetry
What are the types of rotational symmetry?
Cyclic symmetry - single axis of rotation
Dihedral symmetry - n-fold rotation axis intersects a twofold rotation axis at right angles
What does protein stability depend on?
Hydrophobic effect Electrostatic interactions Hydrogen bonding Di-sulphide bridges Metal ions
These stabilising forces work against destabilising (entropy)
Why are proteins deemed relatively stable?
Energy needed to denature (100 residue protein) 40 kJ mol-1
Energy required to break H bonds = 20 kJ mol-1
What is the hydrophobic effect?
Causes non-polar substances to minimise their contacts with water
= aggregation of non-polar side chain amino acids in the interior of the protein
What electrostatic interactions contribute to protein stability?
Van der Waals Hydrogen bonds (not very effective) - they fine tune the tertiary structure
Ion pairs/salt bridge - electrostatic interactions between oppositely charged side chains e.g. Lys and Asp
Describe the help of disulphide bridged?
They cross-link extracellular proteins i.e. within and between polypeptide chains
What do metal ions do?
They stabilise some small domains - internally cross-link proteins
Example: Zinc fingers
1 or 2 Zn2+ ions are tetrahedrally coordinated by side chains of Cys, His
(usuful as it only has 1 stable oxidation state = no undergoing redox)
What can denature proteins?
Temperature - alters optical rotation, viscosity + UV absorption
pH - alters ionisation states, changing protein charge distributions
Detergents - associate with nonpolar residues = interferes with hydrophobic interactions
Chaotropic agents
What are chaotropic agents?
Ions or small organic molecules that increase the solubility of nonpolar substances in water
e.g. Guanidinium ion and urea
Give an example of how a denatured protein can be renatured?
RNase A (4 disulphide bonds) Denatured using urea and mercaptoethanol
Removing these and supplying O2 = spontaneous renaturisation
What property of proteins allows us to monitor them?
They are dynamic = flexible and rapidly fluctuating
We can monitor:
bond vibrations, bond rotations, loop movements and side/main chain dynamics
What pathway does protein folding follow?
An efficient folding pathway from high energy and high entropy to low energy and low entropy (AKA folding funnel)
They fold in a hierarchical manner
What enzyme acts during protein folding?
Protein disulfide isomerase (PDI) catalyzes disulfide bond formation
