Download Chapter 3: Amino Acids and Peptides

Chapter 3: Amino Acids and Peptides
BINF 6101/8101, Spring 2017
Protein Sequence
Outline
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Amino Acids
q  Proteins are linear polymers of amino acids connected by peptide bonds –
amino acids are the building blocks of proteins
q  There are 20 standard amino acids. Asparagine was first found in 1806 and
the last amino acid discovered (Threonine) was in 1938 (over 130 years
later!!)
q  All 20 amino acids share common structural features: α-amino acids
--each has a carboxyl group and an amino group bonded to the same α-carbon
--differ in R group or side chain
General Structure of an Amino Acid
The twenty α-amino acids that are encoded by the genetic code share the generic
structure, differing only at the R substituent, except for one amino acid
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Stereoisomerism in α-amino Acids
Enantiomers (mirror images)
--All amino acids are chiral
(except glycine)
--Proteins only contain L
amino acids
Perspective
Fischer
projection
Stereoisomerism in α-amino Acids
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Important Terms
q  Hydrophobic: tending to avoid an aqueous environment. Hydrophobic
molecules are non-polar and uncharged. Amino acids with this property are
usually buried within the hydrophobic core of the protein.
Aliphatic: carbon atoms are joined together in straight or branched open
chains rather than in rings.
Aromatic: contains an aromatic ring system.
q Hydrophilic: tending to interact with water. Hydrophilic molecules are polar
and charged. Generally found on protein surface and exposed to aqueous
environment.
Hydrophobic
core
Hydrophilic
surface
These amino acid side chains absorb UV light at 270–280 nm
Amino Acids
Amino Acids
3-Letter
1-Letter
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Ala
Arg
Asn
Asp
Cys
Glu
Gln
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
A
R
N
D
C
E
Q
G
H
I
L
K
M
F
P
S
T
W
Y
V
Carboxyl: D, E
Hydroxyl: S, T
Sulfhydryl: C
Guanidine: R
Imidazole: H
Amido: N, Q
The Classification is Actually More Complicated
M.J. Betts, R.B. Russell. (2003) “Amino acid properties and consequences of substitutions”
Essential Amino Acids for Humans
Question: How many amino acid types we need to get from diet?
(In other words, what are the amino acids that cannot be synthesized by our body?)
•  Cannot be synthesized by humans
•  Must be provided in diet
•  Bacteria and plants can synthesize all 20 amino acids
Histidine (H)
Isoleucine (I)
Leucine (L)
Lysine (K)
Methionine (M)
Phenylalanine (F)
Threonine (T)
Tryptophan (W)
Valine (V)
“KVWaIT FILM”
Atom Naming for Amino Acids
C
CA
CB
CG
CD
CE
O, OXT
ζ
7
N
NZ
ζ
NZ
Atom Naming in PDB
PDB: Protein Data Bank http://www.rcsb.org/pdb/home/home.do
atomic symbol remoteness indicator branch designator
C, N, O, S
Greek letters
"A" for alpha,
"B" for beta,
"G" for gamma,
"D" for delta,
"E" for epsilon,
"Z" for zeta, and
"H" for eta.
1, 2, 3…
PDB: Protein Data Bank
http://www.rcsb.org/pdb/home/home.do
26.173
26.169
25.363
25.215
27.626
28.434
27.465
24.795
23.972
24.700
25.140
22.668
21.675
20.419
19.779
19.003
24.698
25.371
0.149
1.597
1.875
0.973
1.981
0.721
-0.440
3.073
3.469
4.597
5.536
4.145
3.360
4.220
4.628
5.891
4.389
5.384
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
28.61
27.50
26.69
26.48
28.55
29.65
28.77
22.80
22.07
18.49
17.98
24.58
36.59
48.23
53.43
57.07
17.98
17.19
N
C
C
O
C
C
C
N
C
C
O
C
C
C
C
N
N
C
B-factor
(aka Temp factor)
Atom type
Residue name
22.126
21.848
20.582
19.724
21.874
21.899
21.761
20.499
19.360
18.610
19.262
19.669
20.495
20.652
19.341
19.502
17.319
16.468
Occupancy
Atom name
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
4
Z-coordinate
Atom number
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Y-coordinate
PRO
PRO
PRO
PRO
PRO
PRO
PRO
LYS
LYS
LYS
LYS
LYS
LYS
LYS
LYS
LYS
ALA
ALA
X-coordinate
N
CA
C
O
CB
CG
CD
N
CA
C
O
CB
CG
CD
CE
NZ
N
CA
Residue number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Chain ID
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
Record name
The PDB File Format
Numbers are used to discriminate between similar positions…
CB
CB
CB
CG
CD1
CG2
CD2
CG
OG1
OD1
ND2
Here are some harder examples…
CB
CB
CD1
CG
CD2
NE2
ND1
CE1
CE1
CG
CZ
CB
CD2
CD2
CG
CE2
CD1
OH
NE1
CE2
CE3
CZ3
CH2
CZ2
Side-chain Torsion Angles
- With the exception of Ala and Gly, all sidechains also
have torsion angles.
- To do on your own:
-  Count the # of chi’s in each amino acid.
-  Determine why Ala doesn’t have a chi angle.
Chi1: N-CA-CB-CG
Chi2: CA-CB-CG-CD
Chi3: CB-CG-CD-CE
Chi4: CG-CD-CE-NZ
Side Chain Torsion Angles
Number of χ
No χ
Amino Acid Types
Gly, Ala
Only one χ1
Cys, Ser, Thr, Val
Asn, Asp, His, Ile, Leu, Phe, Pro, Tyr, Trp
χ1 ,χ2
Gln, Glu, Met
χ1 ,χ2, χ3
χ1 ,χ2, χ3 ,χ4
Lys
χ1 ,χ2, χ3 ,χ4, χ5
Arg
Take this amino acid as an example
Side Chain Torsion Angles
q  The different conformations of the
sidechain as a function of χ1are referred
to as gauche(+), trans and gauche(-).
q  The amino acid is viewed along the
Cβ-Cα bond
www.cryst.bbk.ac.uk
In case you forgot…..
CH3-CH2-CH2-CH3
Newman projections
Side Chain Torsion Angles
q  It has been shown in the 70s by Janin et al. that different side-chain
conformations don not have equal distribution over the dihedral angle space.
Rather they tend to cluster at specific regions of the space.
Janin J, Wodak S. “Conformation of amino acid side-chains in proteins”, J Mol Biol.
1978,125(3):357-86
Side Chain Torsion Angles
Distribution of side-chain torsion angles for 6,638 leucine residues (403 crystal
structures). The two major rotamers are labeled "1" and "2“.
G.J. Kleywegt and T.A. Jones, Acta Cryst. D54, 1119-1131 (1998).
Right image from Dunbrack’s lab
Side Chain Torsion Angles
http://dunbrack.fccc.edu/
Proline: Side Chain Torsion Angles
http://dunbrack.fccc.edu/bbdep2010/figures/pro0_x1.gif
Side Chain Rotamers
q  Rotamer: Rotational isomer
q  Rotamers are generally defined as low energy side-chain
conformations.
q  Rotamers are knowledge-based. They are derived from
statistical analysis of sidechain conformations in known protein
structures through clustering observed conformations or by
dividing torsion angle space into bins and determining an average
conformation in each bin
q Rotamer libraries: collections of rotamers for each residue type.
In general, rotamer libraries contain information about both the
conformation and the frequency of a certain conformation. There
are several different types of rotamer libraies.
Side Chain Rotamers
http://www.cgl.ucsf.edu/chimera/docs/ContributedSoftware/rotamers/rotamers.html
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Modified Amino Acids Found in Proteins
Reversible Modifications of Amino Acids
Other Uncommon Amino Acids
They are not encoded within the genetic code and not incorporated into
proteins. They are intermediates in the biosynthesis of arginine and in the
urea cycle.
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Titration of Glycine
Cation à Zwitterion à Anion
At acidic pH, the carboxyl
group is protonated and the
amino acid is in the cationic
form.
At neutral pH, the carboxyl
group is deprotonated but the
amino group is protonated.
The net charge is zero; such
ions are called Zwitterions.
At alkaline pH, the amino
group is neutral –NH2 and the
amino acid is in the anionic
form.
Titration Curves for Glutamate
PI: isoelectric point
Net charge is 0. PI for glutamate: 3.22
Titration Curves for Histidine
Amino Acid Sidechain pKa Values
ResiduepKavalues:
CT:3.8(R-CO2H)
Asp:4.0(R-CO2H)
Glu:4.4(R-CO2H)
His:6.5(imidazole)
NT:8.0(R-NH3+)
Cys:8.5(R-SH)
Tyr:10.0(Ph-OH)
Lys:10.0(R-NH3+)
Arg:12.0(guanidinium)
The model pKa for a particular
amino acid residue is determined
for the case when the titratable
group is completely accessible to
the solvent and minimally
perturbed by the surrounding
environment
From: Protein Sci. 2006 May; 15(5): 1214–1218.
Effect of the Chemical Environment on pKa
α-carboxy group is much more acidic than that in carboxylic acids
α-amino group is slightly less basic than in amines
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Levels of Structure in Proteins
Primary structure = the complete set of covalent bonds within a protein
Polypeptides
Linear arrangement of n amino acid residues linked by peptide bonds.
Polymers composed of two, three, a few, and many amino acid residues
are called as dipeptides, tripeptides, oligopeptides and polypeptides.
Proteins are molecules that consist of one or more polypeptide chains.
Formation of a Peptide
Amino Acid 1
Amino Acid 2
Peptide bond
Note: this chemistry will not work as drawn!
Nucleophilic Attack Reactions
The electronegative nucleophile attacks the electropositive center
Nucleophilic Attack Reactions
Reality is more complicated b/c OH- is a horrible leaving group
Formation of a Peptide
Amino Acid 1
Amino Acid 2
Note: this
chemistry will not
work as drawn!
Why is this
important to
biology?
Peptide bond
Peptide bond is the amide linkage that is formed between two amino
acids, which results in (net) release of a molecule of water (H2O).
The four atoms in the brown box form a rigid planar unit and, as we will
see next, there is no rotation around the C-N bond.
A Pentapeptide
Numbering (and naming) starts from the amino terminus
The Peptide Bond
The peptide bond has a partial double bond
character, estimated at 40% under typical
conditions. It is this fact that makes the
peptide bond planar and rigid.
A quick aside…
..
+
+
+
A horrible leaving group
..
+
+
+
A viable leaving group
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Overall amino acid structure
Amino acid stereochemistry
Amino acid sidechain structure & classification
‘Non-standard’ amino acids
Amino acid ionization
Formation of the peptide bond
Disulfide bonds
Disulfide Bond and Insulin
Disulfide Bond in Ribonuclease
Sequence alignment and sequence motif
Protein Sequence Alignment
A multiple sequence alignment:
-CAPSRPLNENDDGR-QAFELIGTAVNM...
-CVPGRGEMEHDD-RDQVLELFGTVVNL...
-AVPKRAALQNDDGR-QGWELYGTVSAQ...
-AVPTKMNCFNDDGR-QSVNLIGTVSGN...
-ILPARTSMCNDDGR-QTIEMKGTPAGG...
--APGK--NGHKLV--Q-FELKGTYSRT...
AFAPRRIKMVNKLGR-QNFTLLGTFERT...
AYRPDRCNTCNKLGR-QDVELMGTDART...
-YRPEEWFGENKLGR-QSAELIGTDERS...
--APL-ETYWPKLGR-QTGALAGTNSAV...
--RPY-KAGWNKLGR-QSYELGGTNPYI...
---PARAKNMG---R-QSYHL--TMEWQ...
Protein structure is more conserved
than protein sequence!
Chothia & Lesk. EMBO J. 5:823-826 (1986).
AN EXAMPLE MULTIPLE SEQUENCE ALIGNMENT.
Conserved residues are indicated by color. Note that gaps tend to cluster together.
Also gaps at the N- and C-terminal ends are more common. Why?
Consensus Sequence and Sequence Logo
P loop, an ATP binding structure
EF hand, a Ca2+ binding structure
Regular expressions provide a coarse-grain summary of an alignment segment.
Sequence logos essentially do the same, but without information loss
(cf. http://en.wikipedia.org/wiki/Sequence_logo).
Signatures of Heme Binding Proteins
32 heme c binding proteins
18 heme b binding proteins