-
Primary structure
description of covalent bonds between amino acids
-
secondary structure
- local spatial arrangement of main chain atoms
- rotation around phi and psi bonds
-
tertiary structure
polypeptide chain, beta and alpha sheets together
-
importance of amino acid sequence
- determines 3-D structure
- reveals structural and functional relationships between proteins
- evolutionary relationship
- provides a partial molecular definition of some genetic disease
-
Sanger sequencing method for amino acids
- 1. Determine N terminus
- 2. Cleave disulfide bonds
- 3.Cleavage
- 4. Sequence fragments
- 5. Line up fragments
-
How to determine N-Terminus
- add FDNP-bond to first amino acid
- strong acid- to remove first amino acid
-
cleavage methods
- trypsin-cuts Coo- side of + amino acid residues: lysine and arginine
- Chymotrpsin: tyrosine, tryptophan phenylalanine
- CNBr: cuts the carboxyl group of methionine
-
sequence amino acids
- Edman Degredation
- 1.Phenylisothiocyanate + peptide
- 2.add base to connect
- 3. add acid to remove first amino acid in the fragment
-
phi bond
amino and alpha carbon
-
psi bond
alpha carbon and carboxyl carbon
-
stability factors involved in alpha helix
- maximum h- bonding
- rigidity of peptide bonds
- side chain interactions
- prolines(creates kink in structure)
-
stability factors involved in beta sheets
- inter or intrachain H-bonding
- small amino acid side chains (in fibrous proteins)
- parallel and anti parallel
-
Beta bend
- 4 amino acids
- to types of bends
- bond between hydrogen(N) and oxygen
-
alpha keratin
- hooves, horns, hard and or flexible
- fibrous structure
- 2 chain coiled helix-protofilament-protofibril
- linked by disulfide binds
-
collagen
- most abundant protein
- Gly-Pro-HyPro(hydroxyproline)
- left hand helix
- 3 helix, tropocollagen super helix right hand
- no space in the middle
-
globular protein structures
complex structures, marginally stable
-
stabilization of globular proteins
- hydrophobic forces (important)
- disulfide bonds
- electrostatic forces
- hydrogen bonds
-
structural constraints of tertiary structures
- L-amino acids
- cis and trans configurations of peptide bonds
- steric constraints on phi and psi bonds
- proline is found in 6% if peptide bonds next to proline, at bends
-
lessons from myoglobin
- predicted that secondary structure exist
- very compact
- hydrophobic residues buried
- proline often at bends
-
structural tendencies of beta sheets
- twist in a right handed fashion
- often connect 2 antiparallel beta chains
-
super secondary structures
- folds/motifs
- Domains
- Tertiary structures
-
folds/motifs
- simpler structural patterns found repeatedly in globular proteins
- folding pattern with two or more secondary structures
-
domains
- independently stable cluster of amino acids
- sometimes has a discrete function
- proteins can have multiple domains
-
different types of motifs
- all alpha(serum albumin)
- alpha/beta (alcohol dehydrogenase)
- all beta (UDP, collagenase)
- alpha+ beta (GFP)
-
protein folding is...
not a random process
-
probable steps of protein folding
- stepwise path (short stretches of secondary structure)
- formation of loose "molten globule" intermediate (enzymers catalyze some steps)
-
what is heme
a porphoryn ring (Pyrole ring) with a Fe +2
-
equilibrium association constant
[PL]/[P][L]=1/Kd
-
binding sites occupied/total binding sites=
[L]/[L]+Kd
-
the lower the Kd
the higher the affinity of the ligand for the protein
-
where is Kd
where [O2] binding sites are half occupies
-
binding equilibirum equation
θ=y/y+x
-
why does o2 bind to heme in globin strucutre more than CO
the distal histidine offers h-bonding which increases its stability
-
myoglobin vs hemoglobin
- hemoglobin multiple subunits (2 with heme) located in blood for O2 transport
- myoglobin 1 unit with heme and located in muscle for storage
-
what conformational change does O2 induce in hemoglobin
- T state tense low afinity for O2, predominate in the absence of O2
- R state relaxed high O2 affinity.
- O2 binding triggers T to R transition and other structural changes
-
regulation of hemoglobin
- hemoglobin transports H+ and CO2(binds to amino teminus) stabilize T state
- binding of 2,3 Biphosphoglycerate stabilize T state
-
enzymes affect...not
rates...equilibrium
-
enzymes...
lower the activation barrier for rxns
-
enzyme principles
- accelerate rxn rates not equilibrium
- catalyzes rxns in both directions
-
two theories of active site function
- lock and key: active site complementary to substrate
- modern: active site complementary to transition state
-
foces that hold enzyme and substrate together
- same as the ones that hold proteins together
- binding energy is substantial (transition state)
-
binding energy
the currency used to reduce activation energy
-
how binding energy is used
- entropy reduction-hold subtrate in proper orientation to react
- desolvation-replace H-bonds to H2O
- strain-facilitate geometric or electrostatic distortion
- induced fit-bring reactive groups on enzyme into proper orientation for catalysis
|
|
