Protein-Derived Amino Acids
Protein - biological polymer of a-amino acids
Simplest protein derived amino acid (aa) - glycine (only achiral
protein-derived aa)
Chapter 24 - Slide 2
Chirality of amino acids
Only one configuration found naturally
- According to R/S system - S
- According to D/L system - L
- put most oxidized carbon at top
- continue carbon chain to bottom
- non-hydrogen substituent on left = L
Natural AA Sidechains (R groups): Non-polar
Chapter 24 - Slide 4
Natural AA Sidechains (R groups): Polar, neutral
Chapter 24 - Slide 5
Natural AA Sidechains (R groups): Polar, acidic
Chapter 24 - Slide 6
Natural AA Sidechains (R groups): Polar, basic
Chapter 24 - Slide 7
Acid-Base Properties of Amino Acids
| Amino Acid | pKa a-CO2H | pKa b-CO2H | pKa a-NH3+ |
|---|---|---|---|
| Aspartic Acid | 2.10 | 3.86 | 9.82 |
Chapter 24 - Slide 8
Significance of pKa
Chapter 24 - Slide 9
Forms of Amino Acid Present as a Function of pH
Chapter 24 - Slide 10
Buffer Regions
Chapter 24 - Slide 11
Challenge:
Tyrosine has an a-CO2H pKa of 2.20, and a-NH3+ pKa of 9.11, and a
sidechain pKa of 10.07 - sketch a titration curve and give the structure
of the major form or forms present at all equivalence and half-equivalence
points.
Chapter 24 - Slide 12
Isoelectric Point
Isoelectric point (pI) - pH at which molecule has no net charge
(find by averaging pkaÕs on either side of neutral form)
Chapter 24 - Slide 13
Isoelectric Precipitation
Amino acids with net charges repel each other and do not easily crystallize from solution - solubility is lowest at the isoelectric point
Chapter 24 - Slide 14
Electrophoresis
A method for separating charged compounds in an electric field
Chapter 24 - Slide 15
Nutrition
Dietary protein provides aaÕs not made in our systems - these are
called
essential
| Essential Amino Acids | Nonessential Amino Acids |
|---|---|
| arginine | glycine |
| histidine | alanine |
| lysine | cysteine |
| isoleucine | aspartic acid |
| leucine | glutamic acid |
| valine | asparagine |
| threonine | glutamine |
| methionine | proline |
| phenylalanine | tyrosine|
| tryptophan | serine |
Polypeptides and Proteins
Proteins and polypeptides are chains of aa's
Dipeptide - 2 amino acidsTripeptide - 3 amino acids ...
Polypeptides have 10 or more amino acids
Proteins have 100s or more amino acids
Chapter 24 - Slide 17
Amino Acid Connections
Chapter 24 - Slide 18
Peptide Bond Geometry
Peptide bonds are planar due to conjugation of nitrogen lone pair with
carbonyl
Peptide bond movie
Chapter 24 - Slide 19
Protein Structural Descriptions
- Primary Structure
- sequence of amino acids
- Secondary Structure
- local three-dimensional patterns
- Tertiary Structure
- three-dimensional shape of an amino acid chain
- Quaternary Structure
- three-dimensional shape of multiple amino acid chains
Primary Structure Determination
Step 1: Amino Acid Analysis
Protein is hydrolyzed in strong acid or base at high temperatures
Mixture of amino acids generated by protein hydrolysis is analyzed by
chromatography
Step 2: Sequence Analysis
protein cleaved into polypeptides
polypeptides subjected to amino acid analysis or Edman
degradation
Chapter 24 - Slide 21
Sample Hydrolysis
Chapter 24 - Slide 22
Sequence Analysis
- A Specific Chemical Cleavage:
- Cyanogen bromide (Br-CN): cleaves peptide bonds formed by carboxyl of methionine
- Specific Enzymatic Cleavages:
- Trypsin: cleaves peptide bonds formed by carboxyl of lysine and arginine
- Chymotrypsin: cleaves peptide bonds formed by carboxyl of phenylalanine, tyrosine, and tryptophan
- Sequential Degradation:
- Edman Degradation: cleaves n-terminal aa
Cyanogen Bromide Cleavage
Chapter 24 - Slide 24
Edman Degradation
This process can be repeated ~20-30 times
Long polypeptides require cleavage to small units which can be
individually
sequenced
Chapter 24 - Slide 25
Challenge
Given:
What is the structure of the original peptide?
Chapter 24 - Slide 26
Secondary Structure
Common local conformations:
- Alpha Helix
- Beta-Pleated Sheet
Stabilized by:
- Hydrogen bonding
- R-groups provided space
- s-trans amide bonds
Alpha-Helix
alpha helix movieChapter 24 - Slide 28
Beta-Pleated Sheet
beta sheet movieChapter 24 - Slide 29
Tertiary Structure
Stabilized by:
- Clustering of hydrophobic sidechains
- Salt bridges - oppositely charged sidechains clustering
- Disulfide bonds - 2 cysteines can be oxidized
Chapter 24 - Slide 30
Quaternary Structure
Forces holding protein chains together:
- Clustering of hydrophobic sidechains
- Disulfide Bonds
Polypeptide Synthesis
Problems:
- 20 different building blocks
- all building blocks connected by amide bonds
- cannot simply mix all needed aa's and allow them to react randomly
Polypeptide Synthesis
- Strategies:
- Linear Synthesis
- Convergent Synthesis
- Solid-Phase Synthesis
- Requirements:
- Protecting groups to prevent unwanted couplings
Linear Polypeptide Synthesis
Chapter 24 - Slide 34
Convergent Polypeptide Synthesis
Chapter 24 - Slide 35
Solid-Phase Polypeptide Synthesis: (Merrifield Synthesis)
- Process:
- Amino-protected amino acid coupled to resin
- Protecting group removed
- Another amino-protected amino acid added
repeat steps 2-3, ending with step 2 - Cleave polypeptide from solid resin
- Advantages:
- Fully automated
- By-products easily rinsed away
Amino Protecting Groups
Amino groups commonly protected as carbamates
Chapter 24 - Slide 37
Carboxyl Protecting Groups
Methyl, ethyl, and benzyl esters
Chapter 24 - Slide 38
Peptide Bond Forming Reaction
Chapter 24 - Slide 39Last modified 12/26/96
Dr. Abby Parrill
Department of Chemistry
Michigan State University
These pages may be downloaded and linked from other pages freely for
academic and educational purposes. Questions, problems, and errors should
be
sent to
parrill@argus.cem.msu.edu.