Chapter 3:

Amino Acids, Peptides, and Proteins

3.1 Amino acids

3.2 Peptides & proteins

3.3 Working with proteins

3.4 The covalent nature of proteins

3.5 Protein sequences & evolution
3.1 Amino acids “2007” Quiz Scores
Amino acids 20 amino acids
share common structural Features.

carbon
 – amino acids
Amino acids can be classified
by R group (side chain).

(K)  one-letter code

    

Greek Greek
Letter Latin  Key Letter Latin  Key

1 Αα Alpha Aa 13 Νν Nu Nn
2 Ββ Beta B b 14 Ξξ Xi Xx
3 Γγ Gamma G g 15 Οο Omicron Oo
4 Δδ Delta Dd 16 Ππ Pi Pp
.
5 Εε Epsilon Ee 17 Ρρ Rho Rr
6 Ζζ Zeta Zz 18 Σσ Sigma Ss
7 Ηη Eta H h 19 Ττ Tau Tt
8 Θθ Theta Qq 20 Υυ Upsilon Uu
9 Ιι Iota I i 21 Φφ Phi Ff
10 Κκ Kappa  K k 22 Χχ Chi Cc
11 Λλ Lambda  L l 23 Ψψ Psi Yy
12 Μμ Mu Mm 24 Ωω Omega Ww
 S R
The "CORN" rule. The groups: COOH, R, NH2 and H are arranged around the chiral
center carbon atom. Sighting with the hydrogen atom away from the viewer,
if arranged clockwise around the carbon atom, then it is the D-form.
If counter-clockwise, it is the L-form.
Amino acid residues in Whereas 9 of the 19 L-amino acids found in
proteins are L stereoisomers proteins are dextrorotatory (at 589 nm).
(Gly, G, 甘 )(Ala, A, (Val, V, 纈 )
丙) pKa ~ 10.0

(Phe, F, 苯丙 ) (Tyr, Y, 酪 ) (Trp, W,

色)
(Leu, L, (Ile, I, 異白 / 異
白/亮) (Met, M, 甲硫亮/ )
蛋)

+ pKa ~ 6.0
+
pKa ~ 8.2 pKa ~ 10.5 pKa ~ 12.8

(Ser, S, 絲 )(Thr, T, 酥 / 蘇(Cys,

) C, 半胱 )(Lys, K, 離 / 賴 ) (Arg, R, 精 ) (His, H,
組)

-
(Pro, P, pKa ~ 3.6 - pKa ~ 4.2
脯)
(Asn, N, 天門冬 醯 (Gln, Q, 麩 / 谷 醯 (Asp, D, 天門冬 )(Glu, E, 麩 / 谷 )
No D/L form! imino acid

(Gly, G, (Ala, A, (Pro, P, (Val, V,

甘) 丙) 脯) 纈)

(Leu, L, 白 / (Ile, I, 異白 / 異 (Met, M, 甲硫 /

亮) 亮) 蛋)
 Amino acid functions other than in peptide

“Insoluble” “Soluble”
drugs or medications Removed from blood in kidney & excreted as urine

“bile salt” One carbon (CH3) carrier

 pKa ~ 8.2

(Ser, S, 絲 ) (Thr, T, 酥 / (Cys, C, 半

蘇) 胱)
 -indole

pKa ~ 10.0

(Phe, F, 苯 (Tyr, Y, (Trp, W, 色 )

酪)
 Amino Acids & Neurotransmitters
A glass of warm milk before bed A cheese omelet in the morning
give a nice sleep at night give a morning lift
(w/ tyramine “an epinephrine
analog”)

If low  Parkinson’s disease

Extreme high If high Schizophrenia disorder
“manic”

Extreme low “Flight & Fight”

hormone
“depression”
w/ sedative effect
 -imidazole
*

H+
+ + pKa ~ 6.0 “a potent vasodilator”
pKa ~ 10.5 pKa ~ 12.8 released as immune respond

(Lys, K, 離 / (Arg, R, 精 ) (His, H,

賴) 組)

*In urea cycle

 * MSG
(monosodium glutamate)
“Chinese restaurant syndrome”
–
pKa ~ 3.6
–
pKa ~ 4.2

(Asp, D, 天門冬 ) (Glu, E, 麩 /

谷)

(Asn, N, 天門冬 醯 (Gln, Q, 麩 / 谷 醯

(Gly, G, 甘 )(Ala, A, (Val, V, 纈 )
丙) pKa ~ 10.0

(Phe, F, 苯丙 ) (Tyr, Y, 酪 ) (Trp, W,

色)
(Leu, L, (Ile, I, 異白 / 異
白/亮) (Met, M, 甲硫亮/ )
蛋)

+ pKa ~ 6.0
+
pKa ~ 8.2 pKa ~ 10.5 pKa ~ 12.8

(Ser, S, 絲 )(Thr, T, 酥 / 蘇(Cys,

) C, 半胱 )(Lys, K, 離 / 賴 ) (Arg, R, 精 ) (His, H,
組)

-
(Pro, P, pKa ~ 3.6 - pKa ~ 4.2
脯)
(Asn, N, 天門冬 醯 (Gln, Q, 麩 / 谷 醯 (Asp, D, 天門冬 )(Glu, E, 麩 / 谷 )
 ~128-18
138 ~2.2 ~9.5
= ~110
Uncommon amino acids also have important functions.

: in collagen

: in collagen

: in myosin

: in prothrombin (binding Ca++)

 -Lys
-Lys

-Lys

-Lys

In elastin:
Intermediates in urea cycle
21st amino acid

(Orn)

Using stop codon “UGA”

& ~300 more in cells.
Amino acids can act as acids & bases.

“amphoteric electrolyte”
(an ampholyte)

H+ H+
R– R– R–

Net charge: +1 0 -1
Amino acids have characteristic titration curves.

Isoelectric point (pH):

pI = ½ (pK1 + pK2)
= ½ (2.34 + 9.60)
= ~5.97
Net charge: +1 +½ 0 -½ -1

Titration curves predict the electric charge of amino acids.

Henderson-Hassebalch equation: pH = pKa + log([A-]/[HA])
Amino acid is more acidic than
corresponding aliphatic carboxylic acid / amine.
Amino acids differ in their acid-base properties.

pI = ½ (pK1 + pKR)
= ½ (2.19 + 4.25)
Net charge: +1+½ 0 -½ -1 -1½ -2 = ~3.22
 pI = ½ (pKR + pK2)
= ½ (6.0 + 9.17)
= ~7.6

Net charge: +2+1½ +1 +½ 0 -½ -1

3.2 Peptides & proteins
Peptides are chains of amino acids.

“amino acid monomer1” “amino acid monomer2”

“monomer” G = (-), while high Ea

vs. t½(half-life) = ~7 yr
“residue” under most
intracellular condition.

“amino acid residue1” “amino acid residue2”

Dipeptide
* Oligopeptide,
e.g., a pentapeptide,

NC
“Amino-terminal residue” “Carboxyl-terminal residue”
“N-terminal residue” “C-terminal residue”
This pentapeptide can be nammed as:
SGYAL, Ser-Gly-Tyr-Ala-Leu,
serine-glycine-tyrosine-alanine-leucine,
serylglycyltyrosylalanylleucine.
* Polypeptide (i.e., Mr < 10,000)
* Protein (i.e., Mr > 10,000)
Peptides can be distinguished by their ionization behaviors.

Peptides / proteins have their characteristic pI.

pKa ~9.5
pH 2.5 4.5 9.5 10.5
Net +2 +1 0 -1 -2
charge

pI = ½ (4.5 + 9.5)
= ~7.0
pKa ~4.5
* While in closed packed environment of a protein,
(such as active site of an enzyme)
their pKa may significantly changed
by nearby +/- charges.

pKa ~2.5 pKa ~10.5

 Some Small Peptides of Physiological Interest
Dipeptide
Phe

Aspartame
(NutraSweet®)

PKU (Phenylketonuria)
Inborn Errors of Metabolism “accumulated”

Aspartame®  Alatame®
( Phe  Ala )
Tripeptide
Glutathione

vs. “cysteine, Cys”

“cystine”
Cys-Cys
CySS
 Pentapeptide Cyclic nonapeptide
Enkephalin
(nature occurring analgesic)
Leucine enkephalin
Tyr – Gly – Gly – Phe – Leu

Methionine enkephalin
Tyr – Gly – Gly – Phe – Met Oxytocin
Opiates (Structural analog)

Vasopressin
The mode of action of an antibiotic
Gramicidin A

NN

Valinomycin
cyclic(D-Val-L-lactate-L-
Val-D-hydroxyisovalerate)3
Biological active peptides & polypeptides
occur in a vast range of sizes. “Multisubunit”

* If ≥ 2 identical subunits  “oligomeric protomer”.

i.e., hemoglobin (22): a tetramer / a dimer of  protomers.
Polypeptides have characteristic amino acid compositions.
Some proteins contain chemical groups other than amino acids.
3.3 Working with proteins
Proteins can be separated & purified from crude extract.
Column chromatography Ion-exchange
chromatography
(i.e., Cation-exchange)
Lower soln. pH to 3.5 …

Fractionation & collected

by fractional collector.
Ion-exchange chromatography: stationary phase
Size-exclusion chromatography
(also called:
gel-filtration / molecular sieve chromatography)

Larger particles come out first!

Size-exclusion chromatography: stationary phase
 [Glc ]n
• Carbohydrate polymer Dextran (Sephadex®) [Branched]
¶ Agarose (Sepharose®) [linear]

[ Gal + Alt ]n

¶ Poly-acrylamide
[CH2=CHCONH2]
[Cross linked]

¶: Also used in the stationary phase of electrophoresis.

Affinity chromatography
 ＞＞＞
＞＞＞

＞＞＞
＞＞＞
* 1.0 activity unit:
The amount of enzyme causing transformation
of 1.0 mol of substrate per minute at 25oC.

Unseparated proteins can be quantified.

Proteins can be separated & characterized by electrophoresis.
* Vertical electrophoresis V (velocity ) Z ( net charge )
 ( mobility )  
i.e., polyacrylamide gel E (el . potential ) f ( frict . coeff )

* Horizon electrophoresis
i.e., agarose gel
* Stained by
Croomassie blue (protein)
ethidium bromide (DNA) … etc.
* Radio-isotope labeled by
i.e., 32P, 35S… etc.
“autoradiography”
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Smaller particles come out first!

( vs. size-exclusion chromatography)
Isoelectric focusing electrophoresis
There are several levels of protein structure.
Super-secondary structure
/ Motif / Fold Domain
Subunits
Residue in space of an
Stable recurring 3D 3D structure of oligo/polymeric
sequence
structural patterns the whole protein protein
in part of a protein w/ prosthetic group

Prosthetic group 
Subunit
3.4 The covalent structure (primary structure) of proteins

F. Sanger
Nobel laureate in chemistry
1958: protein sequence
1980: DNA sequence

“Bovine insulin” The function of a protein

A: 22 aa + B: 31 aa depends on its amino acid sequence.
Sanger reagent

Edman reagent

“2  n”
Short polypeptides are sequenced using automated procedures.
Automated Peptide
Sequenator
10 ~ 40 residues in 30 min.
Using as little as ~10 pmol
Large proteins
must be sequenced
in smaller segments.
* Enzymatic cleavage
by protease:

* chemical cleavage:
 Step 1 To obtain its component amino acids
By heating protein solution in 6M HCl at 100~110℃ for 12~36 Hours

? ?
?

Asp + Asn = Asx

Glu + Gln = Glx Also, Trp destroyed by acid hydrolysis
Step 2 To find out how many polypeptide chains contained
By determine its N- or C- terminal residues
N-terminal residue

Free amino acid C-terminal residue

Step 3a,b To obtain manageable length of fragments
By using selectable partial digestive enzymes or chemicals
* Enzymatic partial digestion * Chemical partial digestion

Cyanogen
bromide
Step 4 To obtain complete sequence
By overlapping known sequences of fragments
from ≧2 partial digestions

The amino acid sequences

of millions of proteins have been determined.
I.

II.

IIIa.

IIIb.

IV.
Amino acid sequences can also be deduced by other methods.
i.e., Automated DNA Sequenator

Amino acid sequences provide important biochemical information.

Matrix-Assisted Laser
Desorption / Ionization
Mass Spectroscopy
(MALDI-MS)
Koichi Tanaka

“Light-absorbing matrix”
The Nobel Prize in Chemistry 2002
"for their development of soft desorption ionisation
methods for mass spectrometric analyses of
biomacromolecules"

Electro-Spray Ionization
Mass Spectroscopy
(ESI-MS)
John B. Fenn
“liquid”  “gas”
 M: mass of protein
M  n2 X X: mass of added groups (proton)
For any peak: ( m / z ) 2 
n2 n2: charge
M  (n2  1) X
For its neighboring peak: ( m / z )1 
(differ by 1 proton) n2  1

Ready to solve n2 & (m / z ) 2  X

n2 
M: (m / z ) 2  (m / z )1

M  n2  (m / z ) 2  X 
“Ladder sequence” or “Random cleavage”:
using enzymatic digest, i.e., carboxypeptidase. or partial acid hydrolysis.
which yield a mixture of peptide fragments
that each differ in length by one amino acid residue.

Tandem Mass Spectroscopy

w/ noble gas
(MS / MS)

If the all protein sequences are random,

 For a 10-residue fragment
1 / 2010 = 1/ 1.0 x 1013 !!
More than enough to identify
the unique protein in proteome.
e.g., an intact cell is introduced...
Small peptides & proteins can be chemically synthesized.

R. Bruce Merrifield
Nobel in chemistry 1984
Solid-phase peptide synthesis CN
 Automated Peptide Synthesizer
Synthesis of proteins of 100 residues in a few days
In reasonable yield (vs. E. coli in 5 sec. of ~100% yield).

* Ligation of synthetic oligopeptide into large protein.

* Point mutation.
* Chemical modification (i.e., in active site of enzyme).
* Frankenstein… etc.
 3.5 Protein sequences & evolution
“Bioinformatics”

103 residues for human

28 invariant among species

: invariant residues : variable residues : conservative substitutions

Protein sequences can elucidate the history of life on earth.
99.9% identical in genome

“Molecular phylogeny”
Using cytochrome c
 Myoglobin (Mb):
Human vs. Sperm whale

There are 25 amino acid

changes, while
diverged 100 million yrs ago.
If the rate are uniform,
one replacement
~every 4 million yrs.
Myoglobin (Mb) Hemoglobin (Hb,
22)

* Evolution origin
Blosum (blocks substitution matrix) table
Analyze thousands of short conserved blocks
(but not the entire protein sequence)
to obtain this Blosum62 table (identical in at least 62% residues)
(with differential ± scoring).

i.e., Hb A (Glu)  Hb S (Val)?

* How about using Blosum90 or Blosum50?

* How about comparing DNA sequence of genes?
* … etc.
Consensus sequences & Sequence logos
eg. http://www.expasy.org/PROSITE/
P loop (an ATP-binding structure of G protein)
Polar-
Basic-
Acidic-
Hydrophobic-
residues
EF hand (a Ca2+-binding structure of calmodulin)

Note: {w}  w “not allowed”

Complexity of sequence comparison:
* Lateral gene transfer:
Rare transfer of a gene or group of genes from one organism to another.
“Genetic recombination”
* Homologous recombination
* Site-specific recombination
* DNA Transposition

“Jumping genes”
first observed in maize
in the 1940s
Nobel in medicine 1983

Using computer programs by sliding one sequence past the other,

in comparing amino acid residues of homologous proteins.
(i.e., residues of similar chemical properties)
* Signature sequence
Certain segments of a protein sequence
may be found in the organisms of one taxonomic group
but not in the other groups.
A signature sequence
in the EF-1a (bacteria) vs. EF-Tu (archaebacteria & eukaryote) protein:

* Amino acid substitution are not always random.

Should be calibrated by biochemistry, physiology & fossil record… etc.
* Some proteins evolve faster than the other,
or change faster within one group of species than the other.
Accordingly, an evolutionary tree based on one protein
may differ from those of a tree based on another protein.
Hence, more sophisticated analysis (21st century frontier science).
An Evolutionary Tree derived
from amino acid sequence comparison of GroEL family proteins.
i.e., provide hydrophobic environment to assist protein folding.

“external nodes” “internal nodes”

an extant species an extinct ancestor species
An Evolutionary Tree based on analyses
of many different protein sequences & additional genomic features.

[Note: - - -  “remain under investigation”]

The absence of a significant alignment score
does not necessarily mean
no evolution relationship exists between two proteins.
 3D structural similarities sometimes
reveal evolutionary relationships
where sequence homology has been wiped away by time.
“Cytochrome c” “hemo-/myo- globin”