Peptides : Primary sequence and

1
Lecture 5
Peptides Primary sequence and experimental
techniques
2
Peptide Bond
3
Peptide Bond Formation
Activated Intermediates
4
Naming Peptides
5
Levels of Protein Structure
Primary structure Determination of sequence
6
Protein Homology
BLOSUM60 Matrix
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Disulfide bonds are part of the primarystructure

• Disulfide Bonds

8
How to synthesize a peptide
Solid phase synthesis Merrifield synthesis
1 polypeptide of a determined length
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Peptide Bond Formation In Vitro

• 1. Attachment to of C-terminal amino acid
  (protected on amino group) to resin
• 2. Removal of protecting group
• 3. Activation of next amino acid
• 4. Peptide bond formation
• 5. Removal of polypeptide from resin
• 6. Purification of polypeptide

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Step 1 Attachment to Resin
t-Boc Amino acid
t-Boc
11
Step 2 Remove Protecting Group
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Step 3 Amino Acid Activation
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Step 4 Peptide Bond Formation
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Step 5 Removal of Polypeptide
HF
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How to determine 1º sequence

• 1.Determine amino acid composition
• 2. Determine identity of N-terminal amino acid(s)
• 3. For large proteins, reduce to conveniently
  sized peptides
• -Proteases
• -Chemical reagents
• 3a. Cleave disulfide bonds

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Overview of Protein Sequencing

• 3b. Subject each peptide to stepwise removal and
  identification of amino acids from one end
• -Edman chemistry
• 4. Determine order of peptides in original
  protein and location of disulfide bonds

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Amino acid composition
Hydrolyze protein with 6M HCl
Separate amino acids via column chromatography
Quantify using ninhydrin reaction
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Reactions

• Used for detection and quantitation
• Ninhydrin reaction with amino acids

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Reactions

• Fluorescamine Reaction

Highly Fluorescent
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Determination of N-Terminus Sanger
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Determination of N-Terminus Sanger
O
Ala
Gly
Leu
C
O
Strong Acid

Ala
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Similar reactions can be done with dansyl
chloride and dabsyl chloride
CH3
SO2Cl
N
N
N
CH3
1.Reaction 2.Hydrolysis

CH3
SO2-NH-Ala
N
other amino acids
N
N
CH3
Highly fluorescentgood for very small quantities
of protein
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Cleaving Disulfide Bonds

• Reduction and alkylation or oxidation

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Cleaving Disulfide Bonds

• Reduction

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Cleaving Disulfide Bonds
O

• Acetylation

Cys
I
C
O
O-
Cys
S
C
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Oxidation of Disulfide Bonds
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Sequencing with Edman Degradation
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Sequencing with Edman Degradation
(mildly acidic)
(remaining peptide)
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Cutting Large Proteins with Proteases (Table 3-7)
O
R
CH
C
C
1
2
O
_______R_______
(Others slowly)
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Peptide Cleavage with CNBr
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Peptide Cleavage with CNBr
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Protein Cleavage with CNBr
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Sequencing a large peptide