Tertiary structure Flashcards
helix-turn-helix super secondary structure
A-T base pair can be recognised by amino acid
H-bonds between A-T at major grooves
coiled coil
alpha-keratin form from amphipathic helices
EF hand
calcium ligand
helix-turn-loop
tertiary structure
folding polypeptide where R-group interact
noncovalent forces/bond
hydrophobic interaction
H-bond
VdW and electrostatic
covalent
disulphide bonds
disrupting protein structure
heat pH ionic denaturing agent proteolytic enzymes UV/oxidative/radiation damage
Disrupting protein structure by heat
20-40 degrees
normal intercellular pH
7.2 +/- 0.4
ionic strength
0.1M KCl
denaturing agents
organic solvent chaotropic agent (urea, guanidinium hydrochloride)
proteolytic enzyme
proteases
Hydrophobic collapse
prime driving force for protein folding
- HP cluster of non-polar amino acids
Pi bond interaction - pi-stack/pi-overlap
- aromatic amino acid only
- mixing of clouds of pi e-
how Pi bonds interaction are disrupted
by heat
how HP collapse is broken
by organic solvent/denaturing agent
H bonds
- involve polar non-charged R group
how H-bonds are broken and exposed
by heat, denaturing agents
exposed H-bonds - disrupted by water
Hydrophobic interactions
HP collapse
Pi bond interaction
VdW interaction
very short range effect
weak electrostatic forces
how VdW interactions are broken
by heat, denaturing agent
electrostatic bonds
ionic interaction - salt bridge
between charged residues - acidic and basic, cysteine and tyrosine(ionisable)
when electrostatic bonds are in effect
anytime unless surrounded by HP interaction
zwitterionic form
protonation state of R group affected by pH
isoelectric point (pl)
pH where side chain has not net charge
pKa of ionising group
pH where 50% ionisation has occurred
Henderson Hasselbasch desciption
relationship between pH, pKa and extent of ionisation of weak acid
Henderson Hasselbasch equation
HA A- + H+
pH = pKa +log([A-]/[HA])
charge of protein determined by
pH
number of each amino acid
type of amino acid with ionisable sidechain
salt bridges, ionic bonds
made/broken by change in pH
result of change in pH for changing electrostatic bonds
causes charge of relevant side chains to appear/disappear
pH in lysosome and that function
4.5 - 5 around
activates activity of lysosomal enzymes
changing ionic strength of changing environment
also has same effect on lysosomes
amino acid use in labs
using 6 His
Ni2+ - NTA resin
EDTA
use of 6 His
to purify protein which can be engineered into gene
use of Ni2+ - NTA resin
insoluble
cobalt can be used as well
use of EDTA
chelation
Elute by EDTA but also competition or low pH (His becomes protonated)
phosphorylation of hydroxyl groups
Ser, Thr, Tyr - important molecular switch
as they have hydroxyl groups
phosphorylation effect
changes charge of R-group
phosphorylation - using aspartate
has similar size and charge to Ser
therefore able to mimic switch permanently so protein kinase can’t be used and can’t be phosphorylated
phosphorylation - using alanine
create unswitchable version
therefore kinase can’t add phosphate onto hydroxyl therefore unswitchable
Anfinsen experiment on Ribonuclease - native form to fully unfolded
add 8M and excess beta-mercaptoethanol
disulphide bonds
important for extracellular protein
inside cell oxidising potential is low therefore need special enzymes usually taken place in ER
example for disulphide bonds
cysteine and cysteine - add oxygen to form cystine
add beta-mercaptoethanol to reverse
reverse from unfolded to native form on ribonuclease
remove urea and beta-ME by dialysis
bubble oxygen = >90% = native form
formation of 1% regain activity
remove beta-ME and bubble oxygen then dialyse out urea
formation of 99% regain activity - native form
add trace amount of beta-ME
remove urea and letting bonds form back
result of Anfinsen experiment of ribonuclease
folded, active form protein has lowest free energy
all info need by protein to fold to this structure is encoded in primary structure
in experiment some proteins require
protein disulphide isomerase (PDIs)
correct conformation achieved by
making and breaking disulphide bonds
examples of ‘chaperone’
