1. Biochemistry Conception, theory, research, and application
——Logic and LIY (Learn It Yourself)
Sheng Zhao (赵晟), Biochemistry and Molecular Department of Medical school in Southeast University
Web: or
QQ /MSN/Skype/gChat:
Mobile: or

2. Chapter 1: Protein Structure and Function Amino acid and peptide

3. As an example: A Cool Protein
Magic Color
Marker of the twenty-first century
Nobel Prize in Chemistry 2008

4. Origin of GFP
Jellyfish, Aequorea victoria in 1962

6. Functions in nature
Found in the Pacific Northwest jellyfish Aquorea victoria
Works as a spontaneous fluorescent protein which transduces the blue chemiluminescence of the aequorin protein into green fluorescent light
Serves as a means of communication
Attract prey
Means of protection

7. Green Fluorescent Protein (GFP)

8. A song for GFP ——by Christopher Dinesh Raj
XFPs
A song for GFP ——by Christopher Dinesh Raj
Oh, fluorescent one!
From the time they clones you
And the time, I first set my eyes upon you,
Something drives us
To do something new with you…
…Only to make you pure.
You have been glorified here,
And stolen there,
Still you shine like an emerald in mother nature’s crown.
Allow me to make a new construct out of you,
Mutate and transform you,
Shine some blue light upon you,
And I am certain my peers will turn green,
Yes, green with envy,
Because MY GFP is brighter.
Labeled Endoplasmatic Reticulum
Florescent Zebra

9. Structure of GFP Primary 236 AA residues Secondary
* 1 β pleated sheet composed of 11 antiparallel strands (48 %)
* 5 α helices (5%)
20 β turns (bends)
2 γ turns
12 β bulges
7 β hairpins
Supersecondary
β barrel
Fluorophore (Chromophore) in center

10. Amino acids Monomeric building blocks of proteins
20 different standard protein amino acids
Same general structure
Differ in side chain (R group)
All organisms have same set of 20
2 rare protein amino acids
selenocysteine and pyrrolysine
Different activities and shapes of proteins due to different amino acid sequences

11. Amino acids Different side-chain (R group)
-Carbon atom
-Amino group
(N-terminal)
- Carboxyl group
(C-terminal)
Different side-chain (R group)
Different chemical and physical properties

12. Properties of Amino Acids
Chirality
Structure
Acid / Base properties
Chemical reactions
Capacity to polymerize

13. 1. Chirality: L-Amino Acids
Since all amino Acids but glycine (R=H) are chiral molecules, it’s possible to have enantiomers/ stereoisomers. However, nature uses only one enantiomer, the L-amino acid.
The two enantiomers of alanine, D-Alanine and L-Alanine

14. 2. Structure: Amino Acid Classification
Aliphatic
Aromatic
Sulfur containing
Polar/uncharged
basic/acidic
Hydrophobic- water fearing.
non-polar side chains
Hydrophilic- water loving. polar, neutral chains negatively charged, positively charged

15. Non-polar Amino Acids
There are 8 non-polar amino acids:

16. Polar, Uncharged Amino Acids
There 7 polar, uncharged amino acids:

17. Polar, Charged Amino Acids
There are 5 polar charged amino acids:

18. 21st & 22nd AAs

19. Groups and Abbreviations of AA
Amino Acid
3-Letter
1-Letter
Side-chain polarity
Side-chain charge (pH 7.4)
Hydropathy index
Absorbance
λmax(nm)
Alanine
Ala A
nonpolar
neutral
1.8
Arginine
Arg R
Basic polar
positive
−4.5
Asparagine
Asn N
polar
−3.5
Aspartic acid
Asp D
acidic polar
negative
Cysteine
Cys C
2.5
250
Glutamic acid
Glu E
Glutamine
Gln Q
Glycine
Gly G
−0.4
Histidine
His H
positive(10%)neutral(90%)
−3.2
211
Isoleucine
Ile I
4.5
Leucine
Leu L
3.8
Lysine
Lys K
−3.9
Methionine
Met M
1.9
Phenylalanine
Phe F
2.8
257, 206, 188
Proline
Pro P
−1.6
Serine
Ser S
−0.8
Threonine
Thr T
−0.7
Tryptophan
Trp W
−0.9
280, 219
Tyrosine
Tyr Y
−1.3
274, 222, 193
Valine
Val V
4.2
21st and 22nd amino acids
3-Letter
1-Letter
Selenocysteine
Sec U
Pyrrolysine
Pyl O
Ambiguous Amino Acids
3-Letter
1-Letter
Asparagine or aspartic acid
Asx B
Glutamine or glutamic acid
Glx Z
Leucine or Isoleucine
Xle J
Unspecified or unknown amino acid
Xaa X
Unk is sometimes used instead of Xaa, but is less standard.

20. Maps of amino acid Amino Acid 3-Letter 1-Letter Alanine Ala A Arginine
Asparagine
Asn N
Aspartic acid
Asp D
Cysteine
Cys C
Glutamic acid
Glu E
Glutamine
Gln Q
Glycine
Gly G
Histidine
His H
Isoleucine
Ile I
Leucine
Leu L
Lysine
Lys K
Methionine
Met M
Phenylalanine
Phe F
Proline
Pro P
Serine
Ser S
Threonine
Thr T
Tryptophan
Trp W
Tyrosine
Tyr Y
Valine
Val V

21. Essential Amino Acids
(*) Essential only in certain cases.
Human body is able to synthesize only 10 of them. The other 10 must be obtained from food (essential amino acids).
Essential
Nonessential
Histidine
Alanine
Isoleucine
Arginine*
Leucine
Asparagine
Lysine
Aspartic acid
Methionine
Cysteine*
Phenylalanine
Glutamic acid
Threonine
Glutamine*
Tryptophan
Glycine
Valine
Ornithine*
Proline*
Selenocysteine*
Serine*
Tyrosine*

22. UV-absorbing Properties of Amino Acids
Only three amino acids, Phe, Tyr, and Trp, absorb light in the near UV range (230 nm-300 nm). These amino acids dominate the UV absorption spectra of proteins. The wavelength maxima for tyrosine and tryptophan are around 280 nm. In contrast, nucleic acids (we will discuss later) have absorption maximum of 260 nm. Thus a simple UV scan can allow one to distinguish between protein and nucleic acids.

23. Amino Acids not found in Proteins
-Amino group
(N-terminal)
- Carboxyl group
(C-terminal)
-Carbon atom

24. 3. Acid / Base properties: Zwitterions
A Zwitterion is a dipolar ion. Since amino acids contain both an acid and a base, an internal acid-base reaction forms a zwitterion.
amino acid
zwitterion
Amino acids exist primarily as zwitterions.

25. Zwitterions
Amino acid zwitterions are amphoteric. They can react as either acids or bases.
In acid solution
zwitterion
protonated
In base solution
zwitterion
deprotonated

26. Isoelectric Points
The isoelectric point of an amino acid occurs at the pH where the amino acid exists as the zwitterion.
protonated
acid solution
low pH
zwitterion
isoelectric point
deprotonated
base solution
high pH

27. Isoelectric Points No net charge Minimum solubility in water
Protein will precipitate out at its isoelectric point
Principle for separating amino acids and peptides based in electrophoresis:
+ charged amino acids move to – electrode
- charged amino acids move to + electrode
0 amino acids at their isoelectric points do not move

28. Electrophoresis
A mixture of histidine, serine, and glutamic acid can be separated by electrophoresis at pH = 5.68. - +
positively charged (protonated) histidine
negatively charged (deprotonated) glutamic acid
serine at its isoelectric point at pH = 5.68

29. Electrophoresis

30. Amino Acid separation by Ion Exchange
Unfortunately, amino acids are not colored as described in this overhead. Therefore, what methods would you use to first check if an amino acid is indeed present?

31. Ion Exchange

32. 4. Chemical reactions Different side-chain (R group)
-Carbon atom
-Amino group
(N-terminal)
- Carboxyl group
(C-terminal)
Different side-chain (R group)
Different chemical and physical properties

33. Reaction with Ninhydrin
Amino group
Reaction with Ninhydrin
Used to visualize spots or bands of amino acids separated by chromatography or electrophoresis. (Qualitatively )
Deep purple color formed with traces of any amino acid except Pro (yellow)
Reaction with Nitrous acid (HNO2 ) and Van Slyke Determination
Nitrogen gas which can be observed qualitatively or measured quantitatively.

34. Reaction with formaldehyde and formol titration
Amino group

35. Amino group and Carboxyl group
A Peptide Bond is an amide bond linking together amino acids forming peptides and proteins.
Amino group and Carboxyl group
peptide bond

36. R-Groups
In solution it is the nature of the amino acid R-groups that dictate structure-function relationships of peptides and proteins.
The hydrophobic amino acids will generally be encountered in the interior of proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are generally found on the exterior of proteins as well as in the active centers of enzymatically active proteins.
The imidazole ring of histidine allows it to act as either a proton donor or acceptor at physiological pH. Hence, it is frequently found in the reactive center of enzymes.
The primary alcohol of serine and threonine as well as the thiol (-SH) of cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis.
Additionally, the thiol of cysteine is able to form a disulfide bond with another cysteine. Disulfide bonding between cysteines in different polypeptide chains of oligomeric proteins plays a crucial role in ordering the structure of complex proteins, e.g. the insulin and its receptor.
Cysteine-SH + HS-Cysteine <=> Cysteine-S-S- Cysteine (Cystine)

37. + OOC CH SH NH HS - CH COO NH OOC CH S NH COO R group reaction -HH
 2 Cysteine become 1 Cystine + -
OOC CH 2 SH + NH 3 HS - CH 2
COO + NH 3
-HH -
OOC CH 2 S + NH 3
COO
Disulfid bond
Cystine

38. Structure of GFP Primary 236 AA residues Secondary
* 1 β pleated sheet composed of 11 antiparallel strands (48 %)
* 5 α helices (5%)
20 β turns (bends)
2 γ turns
12 β bulges
7 β hairpins
Supersecondary
β barrel
Ligand
* Fluorophore (Chromophore)

39. Peptides
Dipeptides: two amino acids linked by peptide bond (amide linkage)
Peptides are written so that the free NH2 group is on the left and the free COOH group is on the right.
N-terminus: alanine
C-terminus: aspartic acid
Ala-Asp
alanylaspartic acid

40. Peptides
Tripeptides
Ser-Cys-Asn
Serinylcysteinylasparagine

41. Properties of Peptides
Acid-base properties——also have pI
Biuret reaction (react with copper sulfate in highly alkaline solutions to produce a violet color. First step of Lowry assay)
Degradation
Biological function

42. Naturally-occurring peptides
Peptides synthesized on the ribosome
(1) coded by their own genes
(2) synthesized in the form of precursor
(3) never contain D-AA
(4) an example: oxytocin
Peptides not synthesized on the ribosome
(1) synthesized by a series of enzymes outside the ribosome
(2) may contain D-AA
(3)an example: glutathione

43. Glutathione GSH Glutamate Cysteine Glycine
-Carbon atom
Antioxidant in vivo to protect DNA

44. glutathione reductase (GSR)
Two status of GSH:
GSH (reduced status)
vs. GSSG (oxidized status)
H2O2
2GSH
NADP+
GSH peroxidase
glutathione reductase (GSR)
2H2O
GSSG
NADPH+H+
Antioxidant in vivo to protect DNA

45. Peptide hormone
Many hormones (e.g. neuropeptide) are oligopeptide or polypeptide
Thyrotropin-releasing hormone
(pyro)Glu His Pro-NH2

46. Polypeptides
Proinsulin

47. Peptides & Proteins Peptides contain 50 or fewer amino acids
Dipeptides contain 2 amino acids.
Tripeptides contain 3 amino acids.
Oligopeptides contain 4~10 amino acids
Polypeptides contain 10~50 amino acids.
Proteins contain greater than 50 amino acids.
An amino acid in the peptides or proteins is
called amino acid residue
Peptide chain and Polypeptide chain