Lecture 5: Proteins 2° and 3° structure

week 3:

Sequencing using Mass Spec

1) purify to homogeneity

2) digest the protein into shorter peptides using protease

3) separate and sequence the resulting fragments using tandem mass spec (MS/MS)

B fragment vs Y fragment

B = N term

Y = C term

How to read peaks corresponding to y-type ions

• we are seeing y-type ions

• if peptide isn’t broken then will have largest mass (right side of graph)

• if next signal is says Gly(57), that means a Glycine was lost

• read amino acid sequence from right to left

peptide bond rotation

no freedom of rotation around peptide bond bc of resonance so can’t rotate a double bond

peptide bonds are ____

planar, there are 6 atoms in a single plane

which bonds can rotate in an amino acids

2 bonds adjacent to alpha C

• phi Φ connects to C-N

• psi ψ connects to C-C

ramachandran plot

• many Φ and ψ angles are forbidden bc of steric clashes

• dark blue areas show allowed angles

Why can Gly residues exist outside of normal regions in Ramachandran plot?

no big side chain to cause steric clash

Why are Pro residues confined to highly restricted areas?

bc the R group is covalently bonded to the backbone nitrogen

a-helix

• C=O and N-H do H-bonding every 4 residues

• no steric clashes

• peptide bonds are planar

• more H-bonds = more stable

• helix is not hollow

• right-handed

• R groups come out

• proline can’t form these

• Glycine can’t form these bc has too much conformational flexibility so wants to be coiled

fibrous proteins summary

• long strands or sheets

• usually a single type of 2° structure and 3° is relatively simple

• insoluble in water bc many hydrophobic amino acid residues

• strength and flexibility

Why does collagen require a Gly at every third position?

Gly is at the very tight junctions between individual a-chains

1st x-ray crystal structure

myoglobin

• made of a-helices

b-sheet

• C=O forms H-bonds with N-H neighbor

• consist of two or more strands

• can be flat or twisted

• no steric clashes + ideal angles

• can be antiparallel or parallel

  • H-bonds in line when antiparallel

• R groups come out

Hydrophobic Effect

biggest contribution to protein stability

fibrous proteins types

• a-keratin

  • dimer of one long a-helix

• collagen

  • trimer of left-handed helices

  • not made of a-helices

globular proteins

• spherical shape

• several types of 2° structure

• ex: myoglobin

intrinsically disordered

• no hydrophobic core

• high density of charged amino acids

• functional promiscuity