Primary Structure and Protein Purification

How do you calculate the number of unique sequences

Where R = 20 Amino Acids and n = sequence positions in peptide

R^n = S

How do you calculate the number of unique structures?

Where n = sequence positions

2^n = U

What is the order of naming peptides?

When amino group is on the left..

• Use root name of first amino acid

  • add “yl”

• Carboxy terminal AA name stays the same

How are peptides classified?

Polymers of AA that are less than 12 residues

How are oligopeptides classified?

Polymers of AA about 12-40 residues

How are polypeptides classified?

Polymers of AA that are greater than 40 residues in length

What is the primary protein structure?

The sequence in which amino acids are linked by peptide bonds in a protein

What are the stereochemistry of R groups from left to right?

down and out, up and in

Why is there no free rotation around a peptide bond?

double bond properties from delocalized electrons

Why is the distance between alpha C almost always 3.8?

trans peptide bonds are energetically favored & structure

Why is there a dipole associated with a peptide bond?

COO- and N+ charge can be delocalized

How is the primary structure read?

the exact sequence of amino acids from the N-terminus (amino end) to the C-terminus (carboxyl end)

Primary structure of proteins have what qualities?

• have 1 or more poly peptide chains

• disulfide linkages

• covalent bonds

What do the presence of disulfide linkages tell you about a protien?

they get excreted (disulfide linkages can only happen outside the cell)

What are important points about amino acid acid composition

• defined as the number of each amino acid in a protein

• hydrolyze protein with strong acid or base to break peptide bonds

• chromatography to determine % amount of AA

• AA composition reflects protein function

What limits the number of rounds of Edman degradation?

• acetylation

• cycle efficiency

• peptide degradation

Define protein sequencing

Determining the order of AA in a protein

What are steps that need to be taken before a protein is sequenced?

1. denature proteins

2. cleave disulfies (beta-mercaptoethanol & Dithiothreitol)

3. stabilize product with I-CH2-COO- for Cys

Edman degradation

• sequential subtractive sequencing

• reagents

  • PTH & Trifluoro acid

  • PTH plucks, TriF removed PTH

• automation - peptide attached to C terminus to insoluble bead

What are the amino acids that trypsin cleaves

Lys or Arg (high specificity)

What are the amino acids that chymotrypsin cleaves?

Phe, Tyr, or Trp (moderate)

What are the amino acids that Staphylococcal protease cleaves?

Glu or Asp

What are the amino acids that Cyanogen bromide (N triple bond C-Br) cleave?

Met (high specificity)

Defina amino acid modification

• modification controls function

e.g. acetylation, formlyate, phosphorylate, add lipids to attach to membranes, replaces Cys with Se to enhance activity, fluorescent

what is amino acid composition?

number of each amino acid in a protien