Protein Structure

Peptide bond

amide bond that joins two amino acids

residue

amino acid units in a protein

Amino acid sequence

N-C (NH3+ to COO-)
order of AA

Sanger protein sequencing

1. hydrolysis of polypeptide into small bits
2. separating bits
3. arranging bits to find the sequence

Fragmentation

Trypsin - Lys, Arg (+) KR
Chymotrypsin -Phe, Tyr, Trp (aromatic) FYW
CNBr - Met (start) M

Secondary structure

H-bonds between C=O & H=N
a-helix
-right handed spiral (1 turn is 3.6 residues)
b-sheet

a-Helix

peptide is planner (cis or tans)
H bond between every 4th aa

disruptions:
-electrostatic repulsion
-bulky R groups
-small R groups (favour b)
-proline

b-Conformation

peptide is planner (cis or trans)
H bond between adjacent strands
-antiparallel 7
-parallel 6.5
small R groups

Tertiary structure

globular form
-hydrophobic core (hydrophobic effect)

stablizing:
-disulfide
-electrostatic/ionic
-h bonds
-metal chelation

Disulfide bonds

crosslink protein with covalent bonds
Cys

Metal chelation

salt bridge to stabilize charges
N(+) |||| Me++ |||| O-

Quaternary structure

interaction of globular polypeptides
-same factors as 3

Protein folding

dependent on aa sequence

Protein denaturation

native protein converted to unfolded
1. heat
2, change in solvent
-few proteins can reverse spontaneously (need chaperons)

Prostheic groups

attached non-amino acids
cofactor
-organic (coenzymes)
-inorganic

Ion exchange chromatography

protein purification
1. beads with sulfonic acid (anionic)
2. AA in buffer at 2
3. AA washed with higher pH
4. pH is pI of AA it will wash away
-salt can remove AA (compete for binding sites)

Size-Exclusion (Gel Sieving) chromatography

1. bead with pores
2. mixture of proteins
Big proteins -quick, can't go through pores
Small proteins -slow, go through beads

Affinity Chromatography

1. beads with ligand
2. mixture of proteins
3. hexokinase binds, others washed
4. ATP added (takes binging site) to unbind hexokinase

SDS-PAGE

1. crosslinked polyacrylamide gel
2. detergent binds to proteins
3. electrical potential moves proteins
4. stained