Transcript Document

Basic protein structure and stability I:
Formation of peptide bonds/
properties of amino acids
Biochem 565, Fall 2008
08/25/08
Proteins are the primary functional
manifestation of the information in genomes
DNA sequence
transcription
RNA sequence
translation
protein
sequence
atgcaaactctttctgaacgcctcaagaagaggcgaattgcgttaaaaatgacgcaaaccgaa
ctggcaaccaaagccggtgttaaacagcaatcaattcaactgattgaagctggagtaaccaa
gcgaccgcgcttcttgtttgagattgctatggcgcttaactgtgatccggtttggttacagtacgg
aactaaacgcggtaaagccgcttaa
augcaaacucuuucugaacgccucaagaagaggcgaauugcguuaaaaaugacgcaaacc
gaacuggcaaccaaagccgguguuaaacagcaaucaauucaacugauugaagcuggagua
accaagcgaccgcgcuucuuguuugagauugcuauggcgcuuaacugugauccgguuug
guuacaguacggaacuaaacgcgguaaagccgcuuaa
MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEAGVT
KRPRFLFEIAMALNCDPVWLQYGTKRGKAA
protein
structure
protein
function
alpha carbon
O
H2N
CH
C
O
OH
H3N
R
O
The zwitterionic form is
the predominant form at
neutral pH
side chain
O
C
a-amino acids-the building blocks
of proteins
C
R
carboxylic acid
group
amino group
CH
H3N
O
C
H
R
The alpha carbon is
a chiral center--natural
proteins are made of
L amino acids (shown
above) as opposed to
D
The protein alphabet--the 20 amino acid R
groups
NH3 

O
O
2
O
2
CH2 
1
HN
C
O
2
1
N

1
C 
CH2 
CH3 
CH2 
CH2 
CH2 
CH2 
H
CH2 
CH 
A
C
E
F
G
H
I
D

H2N
NH2
C
CH3 
S
CH2 
CH2 

1
O
NH2 2
O
C 
C


CH2
HC
a
P
2 H3C
CH2 1
CH 
CH2 
CH2 
2

CH3
CH2 
L
2


1 H3C
K

1
OH
NH
3
NH 
1 HO
CH2 
CH2 
CH2 
OH 
CH2 
CH2 
CH2 
Q
R
S

2
3
CH3 
2
CH 
H2
C

CH2  H2C
N
NH2 2
CH2 
CH3 
SH 

M
2
1

1
CH2 
1 H3C
CH3 
1
1
2

CH2 
CH 
N
T
V
W
Y
Aromatic ring numbering/naming (IUPAC)
2

1 HN
3
7a
H2
C
CH
3a
7

NH2
C
4
1
a
N
2
O
OH
NH2
H2
C
CH
3
C
4
6
OH
5
5
6
4
HO
1
3
2
a
5
HN



NH2
H2
C
CH
a
C
OH
IUPAC nomenclature:
http://www.chem.qmw.ac.uk/iupac/AminoAcid/index.html
O
O
Proteins are made by controlled polymerization
of amino acids
water is eliminated
O
two amino acids
condense to form...
H2 N
CH
O
C
OH
H2 N
CH
R1
OH
R2
N or amino
terminus
H2N
...a dipeptide. If
there are more it
becomes a polypeptide.
Short polypeptide chains
are usually called peptides
while longer ones are called
proteins.
C
O
CH
C
R1
O
NH
CH
C
R2
peptide bond is formed
residue 1
residue 2
C or carboxy
terminus
OH
+ HOH
Solid phase peptide synthesis (SPPS)
P2
P1
O
Fmoc
HN
O
AA2
Fmoc
OH
AA1
HN
Resin
A
activation
deblocking
Fmoc
P2
P1
O
Fmoc
HN
O
AA2
AA1
H2N
Resin
Resin
A
solid support
Fmoc
P1
fmoc protecting group
P2
AA1
AA2
A
A
protecting groups
for side chains
1st and 2nd
amino acids
carbonyl activating
group
P2
P1
O
Fmoc
HN
AA2
repeat steps
for each amino
acid in peptide,
then deblock,
deprotect,
cleave off resin
coupling
O
NH
AA1
Resin
adapted from Sigma-Aldrich website
Solid phase peptide synthesis (SPPS)
P2
P1
O
Fmoc
HN
at the end a final
deblocking is done
followed by removal of
the side-chain
protecting groups and
cleavage from the
resin to recover the
peptide
NH
AA1
Fmoc
final
deblocking
P2
P1
O
AA2
H2N
SPPS using Fmoc can
be used to make
peptides up to 70-100
residues in length
(chemical ligation can
be used to make
longer ones)
AA2
O
Resin
P2
O
NH
AA1
AA2
Resin
deprotection and
cleavage
P1
O
H2N
Resin
O
NH
AA1
OH
Peptide bond formation in vivo
P-site
t-RNA
A-site
NH2
NH2
N
N
N
N
O
O
P
N
O
P
O
H
H
O
H
H
O
OH
H
H
H
H
OH
O
H
O
R1
NH
peptide
N
O
O
O
O
N
N
O
aminoacyl
t-RNA ester
activates
carbonyl, making
peptide bond
formation favorable
O
H2N
adenine 2451
R2
of 23S ribosomal
RNA abstracts proton
from amino group,
catalyzing nucleophilic attack
H
chemical protecting groups
are not necessary because
the ribosomal machinery
ensures selective positioning
and activation of the reactants
Peptide bond formation in vivo
new aminoacyl t-RNA comes into
A-site
peptidyl t-RNA shifts to P-site
P-site
A-site
NH2
NH2
N
N
N
N
O
O
P
N
O
P
H
O
O
O
O
O
N
N
O
H
OH
H
H
O
OH
H
H
H
H
deacylated t-RNA
leaves P-site
O
OH
O
H
N
O
R2
HN
peptide
H
R1
H
N
Properties of the amino acid side chains
•
•
•
•
•
•
size
acid-base equilibria
hydrophobicity/polarity
tautomerism
oxidation/reduction of cysteine
chemical reactivity (next lecture)
Sizes of amino acids
a.a
A
R
D
N
C
E
Q
G
H
I
L
K
vol (Å3)
88.6
173.4
111.1
114.1
108.5
138.4
143.8
60.1
153.2
166.7
166.7
168.6
surface area(Å2) a.a
vol (Å3) surface area(Å2)
M
162.9
185
115
F
189.9
210
225
P
112.7
145
150
S
89.0
115
160
T
116.1
140
135
W
227.8
255
Y
193.6
230
190
V
140.0
155
180
75
195
volume: Zamyatin A Prog Biophys Mol Biol 24, 107 (1972)
surface area: Chothia C J Mol Biol 105, 1 (1975)
175
170
200
Acid-base titration curves of ionizable side chains
base
1
eq.
OHadded
Asp
and
Glu
Cys
His+
Lys+
Tyr
pKa
Arg+
0
3 4 5 6 7 8 9 10 11 12 13 14
physiological pH
pH
acid
The basic side chains
NH2
H2N
NH3
C
NH
CH2
HN
HO
C
CH2
CH2
CH2
CH2
CH2
CH2
CH
NH
CH2
NH2
O
pct occurrence
in proteins
lysine
Lys
K
5.9%
arginine
Arg
R
5.1%
histidine
His
H
2.3%
pKa ~ 10
almost always
positively charged
in proteins
pKa ~ 12
almost always
positively charged
in proteins
pKa ~ 6
means that
often it is
not charged
The acidic side chains
O
O
O
HO
C
O
C
C
CH2
CH2
CH2
CH
and these
carboxylic
acid side chains
are closely
related to their
amide versions...
HO
NH2
NH2
O
C
O
C
C
CH2
CH2
CH2
CH
NH2
O
O
aspartate
Asp
D
5.3%
NH2
glutamate
Glu
E
6.3%
generally negative charged in proteins
because conjugate carboxylic acids have
pKa of about 4
asparagine
Asn
N
4.3%
glutamine
Gln
Q
4.3%
Shifting of side chain titration curves
base
1
NH
H2
C
N
eq.
OHadded
His+
pKa
NH
H2
C
N
H
0
3 4 5 6 7 8 9 10 11 12 13 14
physiological pH
pH
acid
Poorly populated but highly reactive forms of
amino acids
base form of lysine not highly populated in general at
physiological pH, but is a reactive nucleophile, and if
present even in minuscule amounts may do chemistry
H
NH3
NH2
CH2
CH2
R
N
O
– H+
CH2
CH2
CH2
pH 7
CH2
H
N
C
O
CH
CH2
NH
H
N
C
O
H
R
CH2
CH2
CH2
CH2
CH2
CH
NH
HO
C
O
CH
H2O
NH2
Kyte-Doolittle hydropathy of amino-acid residues
side chain
Ile
Val
Leu
Phe
Cys
Met
Ala
Gly
Thr
Trp
hydropathy index
4.5
4.2
3.8
2.8
2.5
1.9
1.8
-0.4
-0.7
-0.9
side chain
Ser
Tyr
Pro
His
Glu
Gln
Asp
Asn
Lys
Arg
hydropathy index
-0.8
-1.3
-1.6
-3.2
-3.5
-3.5
-3.5
-3.5
-3.9
-4.5
Many attempts have been made to quantify polarity, nonpolarity
(hydrophobicity) of amino-acid residues in terms of scales. Kyte-Doolittle is a
classic one. It is based on transfer free energies from nonpolar solvents to
water combined with measurements of the tendency of residues to be buried
in proteins.
nonpolar--blue; polar--red; ambiguous--purple
Kyte J & Doolittle RF J Mol Biol 157, 105-32 (1982)
The aliphatic amino acids (plus methionine)
CH3
CH3
S
CH3
CH3
CH3
H
HO
C
CH
H3C
CH
H3C
H3C
H
CH
CH2
CH2
CH2
CH2
HO
NH2
O
C
NH
O
glycine
Gly
G
7.2%
sometimes
considered
a "polar"
amino acid
alanine
Ala
A
7.8%
the aminoacid equivalent
of vanilla
ice cream
valine
Val
V
6.6%
isoleucine
Ile
I
5.3%
these are branched
at the beta-carbon
leucine
Leu
L
9.1%
the most
common
type in
proteins
proline
Pro
P
5.2%
methionine
Met
M
2.2%
only amino acid with
side-chain fused to
backbone in two places
to make a ring
not aliphatic
because of
sulfur
but is similar
in character
in many ways
(nonaromatic,
nonpolar
residue)
Aromatic side chains
OH
HN
NH
N
CH2
CH2
-O
C
CH
CH2
CH2
NH3+
O
tryptophan
Trp
W
1.4%
phenylalanine
Phe
F
3.9%
both these usually also
considered hydrophobic
amino acids
tyrosine
Tyr
Y
3.2%
histidine
His
H
2.3%
also sometimes
considered an
uncharged polar
residue
often not grouped
with other
aromatics
and also can be
charged/polar
The polar uncharged side chains
OH
NH2
HN
NH2
OH
CH2
HO
C
CH
O
serine
Ser
S
6.8%
HO
H
SH
O
O
N
C
CH2
CH2
CH2
CH3
CH2
C
CH2
CH2
histidine
His
H
2.3%
tyrosine
Tyr
Y
3.2 %
pKa ~ 6
for conjugate
acid means
that sometimes it's
charged in
proteins
not really
very polar
NH2
threonine
Thr
T
5.9%
also has
some
hydrophobic
character
due to methyl
cysteine
Cys
C
6.8%
not as polar
as its sister
serine but
much easier
to ionize to
anion
asparagine
Asn
N
4.3%
glutamine
Gln
Q
4.3%
these two have
a bifunctional
character in the
sense of having
both hydrogen bond
donor and acceptor
groups
pKa ~ 10
means that
it can be
deprotonated
3 (2)
Histidine--the
“ambidextrous”
side chain
 Histidine is just barely
acidic enough to
populate base forms at
neutral pH
 therefore, its base
form is about the
strongest base that can
exist under
physiological conditions
 the base form has two
tautomers: one nitrogen
can act as a base/
nucleophile, while the
other can act as a
hydrogen donor-”ambidextrous”
2 (1)
HN
NH
4 (2)
1 (1)
acid
5 ()
H2C

N
H
O
pKa ~ 7
HN
N
N
NH
base
CH2
H2C
N
H
N
H
O
O
predominant form in model peptides
Cysteine and cystine
Pairs of cysteines frequently undergo oxidation to a disulfide bonded form called “cystine”
disulfide
formation
2
CH2SH
1/2O2
CH2S
2 cysteines
SCH2
H2O
cystine
more hydrophobic than cysteine
disulfide
exchange
R1
S
R2S
SR2
R2
S
R1S
disulfide exchange occurs through the thiolate anion at neutral to basic pH
SR2
Key points about the character of amino
acid side chains
• amino acids don’t fall neatly into classes--they are
different combinations of small/large,
charged/uncharged, polar/nonpolar properties
• how we casually speak of them can affect the way we
think about their behavior. For example, if you think
of Cys as a polar residue, you might be surprised to
find it in the hydrophobic core of a protein unpaired to
any other polar group. But this does happen.
• the properties of a residue type can also vary with
conditions/environment
Grouping the amino acids by properties
from http://www.russell.embl-heidelberg.de/aas/
which adapted it from Livingstone & Barton, CABIOS, 9, 745-756, 1993.