Chem320 Hierarchy of Protein Structure L1

Administration
Email:
hyu@uow.edu.au
Location: 18.G26
Consultation Times:
Tuesday 2:30-4:30pm
Email appointment preferred
References:
Introduction to Protein Structure, Branden & Tooze, 2nd Ed,
Garland Science 1999
Library eReading
Any classical Biochemistry textbooks (Stryer, Lehninger, Voet &
Voet etc.)
Lecture materials and past exam papers.
CHEM320: Bioinformatics
Hierarchy of Protein Structures (L1)
Haibo Yu
School of Chemistry
University of Wollongong
Building 18, Room G26
hyu@uow.edu.au
JWCL281_c05_082-128.qxd
UOW CHEM320 2016, Haibo Yu
6/3/10
10:00 AM
Page 95
Proteins
Why Do We Care?
L1-4: Hierarchy & Classification of Protein Structure

L5: Protein Structure and Function
Dry Prac:
Wet Prac:
Molecular Modelling (Week 1-3)

Protein Chemistry of Lysozyme (Week 4-5)
Section 5-4. Gene Expression and Replication: An Overview
95
Central Dogma of Molecular Biology

Replication
DNA
Transcription
What is the difference between CHEM320 and BIOL213/BIOL214?

Figure 5-20 Autoradiograph of Drosophila melanogaster
DNA. Lysates of D. melanogaster cells that had been cultured
with 3H-labeled thymidine were spread on a glass slide and covered with a photographic emulsion that was developed after a
5-month exposure. The white curve, which resulted from the radioactive decay of the 3H, traces the path of the DNA in this
photographic positive. The DNAs measured contour length is
1.2 cm. [From Kavenoff, R., Klotz, L.C., and Zimm, B.H., Cold
Spring Harbor Symp. Quant. Biol. 38, 4 (1973). Copyright 1974
by Cold Spring Harbor Laboratory Press.]
We focus more on the physical forces underlying protein structure and

function and protein structure classifications.
handling. Before 1960, when this was first realized, the measured molecular masses of DNA were no higher than !10 million D (!15 kb, where
1 kb ! 1 kilobase
pair ! 1000 bp).
3
DNA fragments of uniform molecular mass and as small as a
few hundred base pairs may be generated by shear degrading
DNA in a controlled manner; for instance, by forcing the
Protein
RNA
Translation
Figure 5-21 The central dogma of molecular biology. Solid

arrows indicate the types of genetic information transfers that
occur in all cells. Special transfers are indicated by the dashed
arrows: RNA-directed RNA polymerase is expressed both by
certain RNA viruses and by some plants; RNA-directed DNA
polymerase (reverse transcriptase) is expressed by other RNA
viruses; and DNA directly specifying a protein is unknown but
does not seem beyond the realm of possibility. However, the
missing arrows are information transfers the central dogma postulates never occur: protein specifying either DNA, RNA, or protein. In other words, proteins can only be the recipients of genetic
information. [After Crick, F., Nature 227, 561 (1970).]
Why Do We Care?
Proteins are the most versatile macromolecules in living systems and

serve crucial functions in essentially all biological processes.
Understand the hierarchy of protein structures

(primary, secondary, tertiary and quaternary);
Understand the interactions stabilising protein
structure;
Identify and analyse the various building blocks
in proteins;
Protein engineering - de novo design novel enzymes for

biotechnological applications.
Structure based drug design - they can serve as important drug
targets.
Hierarchy of Protein Structures
1: What Is Primary Structure?
Primary the sequence of amino acids in a protein

Secondary particularly stable local arrangement of amino acid
residues giving rise to recurring structural patterns, e.g. -helix and
-sheet;
Tertiary all aspects of the 3D folding a poly-peptide;
Quaternary When a protein has two or more polypeptide units, their
arrangement in space is referred as Quaternary structure.
Objectives (L1-2)
The linear sequence of amino

acids that composes proteins
(polypeptide sequence);
The linear (1) sequence is
encoded by DNA and transcribed
into mRNA and then translated
into protein;
Each amino acid unit is called
residues;
of the set of 20 in that its side chain is bonded to both

nitrogen
andcalled
thethe ! carbon, linked to an amino group, a
of athe
central
carbon atom,
carboxylic
acid
group,
a
hydrogen
atom, and a distinctive R group. The R
(Figureacid
3.9).consists
Proline markedly influences
protein architecAmino acids are the building blocks of!-carbon
proteins.atoms
An !-amino
group is often referred
to as the side chain. With four different groups conNotation for distinguishing
stereoisomers
ture because
structure
makes
conformationally
restricted
of a central carbon atom, called the ! carbon,
linkeditsto ring
an amino
group,
a it more
nected to the tetrahedral
!-carbon atom, !-amino acids are chiral; the two
The four different substituents of an
mirror-image
forms
are
called
the L isomer and the D isomer (Figure 3.4).
than
the
other
amino
acids.
carboxylic acid group, a hydrogen atom, and a distinctive R group. The R
asymmetric carbon atom are assigned
General
structure
of
amino
acids:
Exception:
group is often referred to as the side chain. With
four different groups cona priority according to atomic number.
H
R:
Side
chain
atoms;
R
H
The lowest-priority substituent,
oftenR
nected to the tetrahedral !-carbon atom, !-amino acids are chiral; the two
hydrogen,
is
pointed
away
from
the
The rest: Backbone
(main
chain)
mirror-image
forms are
called
the atoms;
L isomer and the D isomer (Figure 3.4).
1: Primary Structure - Amino Acids
1: Amino Acids Are Chiral Molecules
The four differen

asymmetric carb
a priority accord
The lowest-prior
hydrogen, is poi
viewer. The con
carbon is called
ter for left, if t
the highest to th
counterclockwis
called R, from th
right, if the pro
viewer. The configuration about

the
C
C
H2
carbon is called S, from
the
Latin
sinisC
CH2
ter for left, ifH2the
from
C progression
CH
+
H
+
NH3 2
NH3
COO
COO
H2C
the highest to the lowest
priority
is
+
N
C
COO
N+
L isomer
D isomer
COO
counterclockwise.
configuration
is
HThe
2
FIGURE 3.9 Cyclic structure of proline.
H2
called R, from
the
Latin
rectus
for
FIGURE 3.4 The
refers chain
to the side
H L and D isomers of amino acids.
TheRside
is chain.
joined to both the !
Prolineright, if theTheprogression
L and D isomersis
areclockwise.
mirror images of each other.
7552dc03_41-76 4/17/01 7:22 AM Page 44
(Pro, P)
carbon and the amino group.
H2
C
Notation for disting
Proline, Pro, P
L: CORN
Only
acids
are constituents
ofalmost
natural
proteins!
OnlyLLamino
amino acids
are constituents
of proteins. For
all amino
acids,
the
L
isomer
has
S
(rather
than
R)
absolute
configuration
(Figure
3.5).
Al+
+
No
satisfactory
explanation
has
been
arrived
at
why
NH3
NH3
COO
COO
though considerable effort has gone into understanding why amino acids in
Three amino acids with relatively simple aromaticamino
side chains
partconfiguration,
of
acidthis
inare
natural
proteins have
this absolute
proteins
have
absolute
no satisfactory
explanation has
e.g.
been
arrived
at.
It
seems
plausible
that
the
selection
of
L over D was arbithe fundamental
as
its
name
indiL isomer
D isomer repertoire (Figure 3.10). Phenylalanine,
configurations.
trary but, once made, was fixed early in evolutionary history.
Glycine
primary
cates,R=H:
contains
a phenyl ring attached in place of one
of the hydrogens of
carboxylic acid
Amino acids in solution at neutral pH exist predominantly as dipolar
FIGURE
3.4group
The L and D isomers of amino acids.R=CH
R refers
to the side chain.
(1)
3: Alanine
amino
alanine.
The
aromatic
ring
of
tyrosine
contains
a
hydroxyl
This hyions
(also group.
called zwitterions).
In the dipolar form, the amino group is protongroup
Question:
#
"Which amino acid is not chiral?
The L and D isomers are mirror images
of each other.
NH
ated (NH
and the carboxyl
3 )chains
droxyl
group is reactive, in contrast with the rather inert
side
of thegroup is deprotonated (COO ). The ionization
state of an amino acid varies with pH (Figure 3.6). In acid solution
9
UOW CHEM320 2016, Haibo
Yu
10
other amino acids discussed thus far. Tryptophan has(e.g.,
anpH
indole
ring group
joined
1), the amino
is protonated (NH3") and the carboxyl group
Only L amino acids are constituents oftoproteins.
For
almost
all
amino
acids,
is
not
dissociated
(COOH).
As the pH is raised, the carboxylic acid is the
a methylene (CH2) group; the indole group comprises two fused rings
first
group
to
give
up
a
proton,
inasmuch as its pKa is near 2. The dipolar
FIGURE 3.5 Onl
the L isomer has S (rather than R) absolute
configuration
3.5). Aland an
NH group. (Figure
Phenylalanine
is purely hydrophobic,
whereas
tyrosine
form persists
until the
pH approaches 9, when the protonated amino group
found in protein
(3)
R
though considerable effort has gone into
understanding
why
amino
acids
in
44
acids have an S a
and tryptophan are less so because of their hydroxyl and NH groups. The
(from the Latin si
proteins have this
absolute
configuration,
no satisfactory
explanation
has
rings of tryptophan
and
tyrosine contain delocalized
! electrons
The counterclockw
1-2
Genes
and aromatic
Proteins
R
R H (4)
R
H
H
H
H
H
arrow from highe
been arrived at. It seems plausible thatthat
the strongly
selectionabsorb
of L over
D was arbiultraviolet
light (Figure 3.11).
substituents indic
H N
COO
COOH
H N
H N
COO
trary but, once made, was fixed early in evolutionary
history.
center is of the S
A
compounds
extinction
coefficient
indicates
its
ability
to
absorb
There
is
a
linear
relationship
between
the
DNA
base
sequence
Hlight. of a
H
mRNA codon -> amino acid
Amino acids in solution at neutralBeers
pH exist
as amino-acid
dipolar
(2)
and the
sequence
of thewavelength:
protein
it encodes
law predominantly
gives gene
the absorbance
(A) of light
at a(1)
given
C
position form, the amino group is protonions (also called zwitterions). In the2nddipolar
The genetic
hereditary
information from
into proteins.
+
Ist position
# code is the formula that converts
COOgenes
NH3
ated (NH3") and the
(COO
).
The
ionU group
C Ais deprotonated
G 3rd(3'position
(5' carboxyl
end)
end)
Every amino acid in a protein is represented by a codon consisting of three
consecutive
Zwitterionic
form
Both groups
deprotonated
Ser with
Tyr
Cys
U
ization state of an amino acidPhevaries
pH (Figure
3.6).
In acid
solution
nucleotides
in the
gene. DNA contains four different nucleotides, with the bases adenine (A),
Phe
Ser
Tyr
Cys
C"
(G), thymidine
(T) and cytosine (C), whose sequence in a gene spells out the sequence
(e.g., pH 1), the amino group Leu
is protonated
(NHA3 ) andguanine
the carboxyl
group
Ser STOP STOP
Leu
Ser STOP Trp
G
of
the
amino
acids
in
the
protein that it specifies: this is the primary structure
of the protein.
Both groups
is not dissociated (COOH). As the pH is raised, the carboxylic acid is the
protonated
Leu
Pro
His
Arg
U
The nucleotide sequence of the DNA is transcribed into messenger RNA (mRNA),
with uridine
first group to give up a proton,
as its pK
near
2. The
dipolar
Leu inasmuch
Pro
His
Arg
C a is (U)
FIGURE 3.6 Ion
FIGURE
3.5
Only
L amino acids
are
replacing
thymine
(T).
Figure
1-4
shows
the
correspondence
between
the
64 possible
Leu
Pro
Gln
Arg
A
function of pH.
form persists until the pH approaches
9, when
the
found
in
proteins.
Almost
all
L amino
Leu
Pro
Gln
Arg
three-baseamino
codons ingroup
mRNA and the
20
naturally
occurring
amino
acids.
Some
amino
acids
G protonated
These groups are important in hydrophobic interactions;
amino acids is alt
2 by as many
4
6 different
8
10
12
14
The zwitterionic fo
acids have
configuration
are specified by only one codon, whereas
othersancanS absolute
be0specified
as six
Ile
Thr
Asn
Ser
U
Glycine
enables
the
peptide
bond
backbone
to
adopt
more
physiological pH.
pH
Ile
Thr
Asn
Ser
C
(fromThere
the Latin
sinister
meaning
left).
codons:
the
genetic
code
is
degenerate.
are
three
codons
that
do
not
code
for
amino
Ile
Thr
Lys
Arg
A
conformations.
Thepolypeptide
counterclockwise
direction
theprocess by which
acids, but signalR the termination of the
chain (stop
codons).ofThe
Met
Thr
LysR Arg
G
R
H+
H+
H
H sequence of the DNA
the nucleotide
is first
transcribed
RNA and then
into acid side chain: Ala scans
arrow
from
to lowest-priority
Val
Ala H Asp
Gly
Alanine
ishighestoften into
considered
as a translated
null amino
U
Val
Ala
Asp
Gly
C
protein
is
outlined
in
Figure
1-5.
substituents
indicates
that
the
chiral
are used to probe structure-function relationships in proteins.
+H N
Val+H Ala
Glu COO
Gly
A
COOH
H2N
COO
3N
3
44
Glycine
(Gly, G)
CHAPTER 3 Protein Structure and Function
Alanine
(Ala, A)
7552dc03_41-76
4/17/01
7:22 AM
Page 44
1: The Genetic Codes Specify 20 Amino

Acid Side Chains
H H
FIGURE 3.7 Structures of glycine and

alanine. (Top) Ball-and-stick models
show the arrangement of atoms and

bonds in space. (Middle) Stereochemically
realistic formulas show the geometrical
Glycine
arrangement of bonds around atoms(Gly,
(seeG)
Chapter 1 Appendix). (Bottom) Fischer
projections show all bonds as being
perpendicular for a simplified
representation (see Chapter 1 Appendix).
H+
Amino
acids
COO
H3N
Glycine
(Gly, G)
Valine
(Val, V)
Leucine H
(Leu, L)
CH
Methionine
(Met, M)
COO
CH3
+H
COO
3N
Alanine
(Ala, A)
HC
CH3
H3C
+H N
3
CH3
CH3
H2C
H2C
CH3
*
C H
Methionine
H
CH2
Isoleucine H
(Ile, I)
COO
+H N
3
Isoleucine
(Ile, I)
H 3N
CH3
H3C
CH3
COO
Alanine
(Ala, A)
Leucine
(Leu, L)
Concentration
U
C
A
G
CH3
Glycine
(Gly, G)
COO
H3N
COO
3N
+H N
3
H H
COO
Valine
( Val, V )

+H
Alanine
(Ala,
C A) COO
+H N
3

arrangement of bonds around atoms (see
CH3
1: AAs with Aliphatic Sidechains

+H N
3
COO
(Met, M)
COO
+H N
3
CH2
+H
COO
3N
CH3
CH3
CH3
Val
Ala
Abbreviations
Glu
Gly
Codons
H+
CH3
centerH isC ofCHthe S configuration.

CH
CH3
CH2
CH2
CH3
CH2

In bacteria and other 11lower organisms,
the
relationship
between
the base sequence
of the gene
C
COO
C
COO
H N
H N
C
COO
H N
H N
C
and the amino acid sequence of the corresponding
protein is strictly
linear: the protein
sequence H
H
H
H
3
Valine
Leucine
Isoleucine
12
COO
Methionine
7552dc03_41-76
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7:22 AM
A Repertoire of 20 Amino Acids
Page 46
JWCL281_c09_278-322.qxd
HC
FIGURE 3.10 Amino acids with

aromatic side chains. Phenylalanine,
tyrosine, and tryptophan have hydrophobic
character. Tyrosine and tryptophan also
have hydrophilic properties because of
their OH and NH groups, respectively.
H O * CH3
C
H
H
CH2
COO
+H N
3
OH
46
COO
+H N
3
2/24/10
1:17 PM
HC
CH2
COO
HN
Page 285
C
C
H
+H N
3
COO
Tyrosine
(Tyr, Y)
Phenylalanine
(Phe, F)
+H N
3
Phenylalanine
(Phe, F)
20,000
10,000
10,000
OH
5,000
A ! !cl
Tryptophan
(Trp, W)
Serine
(Ser, S)
Threonine
(Thr, T)
8,000
4,000
2,000
Trp
6,000
Tyr
0
220
240
Beers law
+H N
3
CH2
COO
CH2
COO
+H N
3
HC
HO
CH
H
C
HN
Threonine
(Thr, T)
Serine
(Ser, S)
CH
CH
Cysteine
(Cys, C)
47
A Repertoire of 20 10,000
Amino Acids
+H N
3
COO
CH3
H
Glutamine
*
(Gln, Q) C H
H
+H N
3
OH
OH
CH2
+H N
3
COO
COO
CH3
+H N
3
COO
Serine
(Ser, S)
Threonine
(Thr, T)
H2N
Extinction coefficient (M1 cm1)
8,000
We turn now to amino acids with very polar side chains that render
them
S
H
highly hydrophilic. Lysine and arginine have relatively long side chains
that
CH2
H Lysine
terminate with groups that are positively charged at neutral pH.
6,000 is
capped by a primary amino group and arginine by a guanidinium
group.
+H N
COO
3
Histidine contains an imidazole group, an aromatic ring that also
can be posCysteine 4,000
itively charged (Figure 3.14).
(Cys, C)
10
SH
CH2
+H N
3
Trp
COO
"1
"1
2,000
Since 1H has an NMR spectrum in a different frequency

range from that of D, the exchange of 1H for D can be readily followed by NMR spectroscopy. Under physiological
conditions, small organic molecules, such as amino acids
and dipeptides, completely exchange their weakly acidic
protons for D in times ranging from milliseconds to seconds.
"1
absorbs light less strongly and at shorter wavelengths.

FIGUREThe
3.13
Structure
cysteine.
absorption
of of
light
at 280 nm can be used to estimate
Tyr
We turn now to amino acids with very polar side chains that render them
0
highly hydrophilic. Lysine and arginine have relatively
chains
220
240long side
260
280 that
300
These residues
and
(nm)
terminate
with groups are
that hydrophilic
are positively charged
atcan
neutralWavelength
pH. Lysine
is
capped by a primary amino group and arginine by a guanidinium group.
form hydrogen bonds (What is a HB?).
Histidine contains an imidazole group, an aromatic ring that also can be positively charged (Figure 3.14).
theC concentration
of a protein
C O of tryptophan and
NH2 in solution if the number
O
tyrosine residues in Othe Cprotein is known. H C
H2C
2
Two amino acids, serine and threonine, contain aliphatic hydroxyl groups
"1
"1
atom fromwhere
a molecule
or a molecular
fragment XH in which X is
! is the extinction coefficient
[in units that are the
reciprocals of molarity and distance in centimeters (M
more electronegative
than
H,
and
an
atom
or a group of atoms in the
cm )], c is the concentration of the absorbing species (in
units of molarity, M), and l is the length through which
same or athedifferent
molecule,
which there is evidence of bond
light passes (in units
of centimeters). in
For tryptophan,
absorption is maximum at 280 nm and the extinction
formation.coefficient is 3400 M cm whereas, for tyrosine, absorption is maximum at 276 nm and the extinction coefInternational
of Pure and Applied Chemistry
ficient is a less-intense
1400 M cm . Union
Phenylalanine
FIGURE 3.13 Structure of cysteine.
HB Acceptor
320
FIGURE 3.11 Absorption spectra of the aromatic amino acids

tryptophan (red) and tyrosine (blue). Only these amino acids
HB
absorb strongly near 280 nm. [Courtesy of Greg Gatto].
FIGURE 3.12 Amino acids containing

aliphatic hydroxyl groups. Serine and
HB Donor
threonine contain hydroxyl groups that

render them hydrophilic. The additional
chiral center in threonine is indicated by
an asterisk.
15
10
FIGURE 3.11 Absorption spectra of the aromatic amino acids

15
tryptophan (red) and tyrosine (blue). Only these amino acids
190
absorb
280 nm.
Gatto].
200 strongly
220 near
240
260[Courtesy
280of Greg
300
320
(nm)
XH ' D2 O XD ' HOD
"1
O
HAsparagine
CHN)
2
(Asn,
H
320
What is a Hydrogen Bond?
1: Polar and Uncharged R Groups
Glutamate
(Glu, E)
20
300
20
Phe
50
HC
C
C
C
make them much more hydrophilic (water loving) and reactive than alanineHC C C CH2
C
CH2
CH2
H
FIGURE 3.10 Amino acids with
H
H
and valine. Threonine, like isoleucine, contains
anchains.
additional
asymmetric
aromatic side
Phenylalanine,
13
UOW CHEM320
14
+H N
+H N 2016,
+H N
C
COO
C Haibo
COO Yu
C
COO
c. Pulsed H/D Exchange Provides Structural Details
3
3
3
tyrosine,
and
tryptophan
have
hydrophobic
center; again only one isomer is present in proteins.
on How Proteins Fold
character. Tyrosine and tryptophan also
H
H
H
Pulsed H/D exchange, a method devised by Walter EngCysteine is structurally similar to
serine
contains
a sulfhydryl,
or thiol
havebut
hydrophilic
properties
because of
lander and Robert Baldwin, is the only known technique
Tryptophan
Phenylalanine
Tyrosine
SH
(Trp, W)
(Phe, F)
(Tyr, Y)
their OH and NH groups, respectively.
S
that can follow the time course of individual residues in a
(SH), group
H in place of the hydroxyl (OH) group (Figure 3.13). The
folding protein. Weakly acidic protons ( H), such as those
CH
2
sulfhydryl group
Pairs of sulfhydryl groups may
CHis
2 much more reactive.
of amine and hydroxyl groups (XH) , exchange with
H
those of water, a process known as hydrogen exchange that
come together to form disulfide

which
are
particularly
important
in
+H N bonds,
C
COO
3
be demonstrated with the use of deuterated water
The hydrogen bondA !is!cl an attractive
interactioncan
between a hydrogen
+H N
COO
[D O; deuterium (D or H) is a stable isotope of H]:
stabilizing
as will be discussed shortly.
3 some proteins,
Beers law
UOW7:22
CHEM320
Haibo
4/17/01
AM 2016,
Page
47 Yu
e 49
100
in this spectral region (ranging into the tens of thousands;

Fig. 9-10). However, polypeptides do not absorb visible
light (! " 400 to 800 nm), so that they are colorless.
For chiral molecules such as proteins, has different values for left and right circularly polarized light, L and R.
The variation with ! of the difference in these quantities,
# " L $ R, constitutes the circular dichroism (CD) spectrum of the solute of interest (for nonchiral molecules L " eR
and hence they have no CD spectrum). In proteins, % helices, & sheets, and random coils exhibit characteristic CD
spectra (Fig. 9-11). Hence the CD spectrum of a polypeptide provides a rough estimate of its secondary structure.
COO
HC
CH
200
CH2
+H N
3
H
C
H
C
500
Section 9-
280 nm
Figure 9-10 UV absorbance spectra of the three aromatic

amino acids, phenylalanine, tryptophan, and tyrosine. Note that
the molar absorbance, , is displayed on a log scale. [After
Wetlaufer, D.B., Adv. Prot. Chem. 7, 310 (1962).]
HN
1,000
280
2,000
Wavelength (nm)
41-76
260
extinction coefficient (M-1 cm-1)
Extinction coefficient (M1 cm1)
C
H
CH3
O
H
+H N
CH
2
C
COO
3
H
COO
where ! is the extinction coefficient [in units that are the

reciprocals of molarity and distance in centimeters 15
(M"1
cm"1)], c is the concentration of the absorbing species (in
units of molarity, M), and Trp
l is the length through which
10
the light passes (in units of centimeters). For tryptophan,
absorption is maximum at 280 nm and the extinction
"1
"1
coefficient is 3400 M cmTyr whereas, for tyrosine,
5 absorption is maximum at 276 nm and the extinction coef"1
"1
ficient is a less-intense 1400 M cm . Phenylalanine
0
absorbs light less strongly and at shorter wavelengths.
The absorption of light at 280 nm can be used to estimate
5

The tendency of hydrocarbons (or of lipophilic hydrocarbon-like
H O * CH
C aliphatic hydroxyl groups. Serine and
H N
C
COO
H
H
groups in solutes) to form intermolecular
aggregates
in an aqueous
276 nm
H
H
H N
COO
H N
COO
medium, and analogous intramolecular interactions. The name arises
an asterisk.
OH
OH
from the attribution of the phenomenon to the
apparent repulsion
240-265 nm
CH
CH
H
C
between water and
hydrocarbons.
the
phenomenon
the concentration
of a proteinHowever,
in solution if the
number
of tryptophan andought
aliphatic
hydroxyl
groups.
Serine
and
tyrosine residues in the protein is known.H N C COO
H N
C
COO
to be attributed to
the effect of the hydrocarbon-like groups
on
the
Two amino acids, serine and threonine, contain aliphatic hydroxyl groups
H
H
(Figure 3.12). Serine can be thought of as a hydroxylated version of alanine,
water-water interaction.
Threonine
Serine
whereas threonine resembles valine with a hydroxyl
group in place of one(Thr, T)
(Ser, S)
an asterisk.
These
AAs have hydrophobic character;
of the valine methyl groups. The hydroxyl groups on serine and threonine
Tyr and Trp also have some hydrophilic character;
make them much more hydrophilic (water loving) and reactive than alanine
Union
of contains
Pure
and
Applied
Chemistry
andInternational
valine. Threonine,the
like
isoleucine,
an additional
asymmetric
concentration
of a protein
in solution
if the number of tryptophan andThey can be especially effective in forming interactions with other flat
center; again only one isomer
is present
tyrosine
residuesininproteins.
the protein is known.
Cysteine is structurally similar
to serine
but serine
contains
a sulfhydryl,
thiol aliphatic hydroxyl groupsmolecules (e.g. protein-ligand interactions);
Two amino
acids,
and
threonine, or
contain
(SH), group in place (Figure
of the hydroxyl
(OH)
(Figure
The
3.12). Serine cangroup
be thought
of 3.13).
as a hydroxylated
version of alanine,
Account for most of the ultraviolet absorbance by proteins; Can be
sulfhydryl group is much
more
reactive.
Pairs
of
sulfhydryl
groups
may
whereas threonine resembles valine with a hydroxyl group in place of one
come together to form disulfide bonds, which are particularly important in
of the valine methyl groups. The hydroxyl groups on serine and threonineused to measure the protein concentrations [Wet Prac];
stabilizing some proteins, as will be discussed shortly.
CH2
CH2
C
H
Tyrosine
(Tyr, Y)
40,000
Tryptophan
(Trp, W)
C
H
CH2
1: AAs with Aromatic Sidechains
What is Hydrophobic Interaction?

H
C
C
H
+H N
3
16
Figure 9-11 C
Polypeptides in
conformations w
of known X-ray
absorption spec
a small differenc
Wetlaufer, D.B.,
Proteins bear n
those of its bac
are engaged i
solvent and, m
protein are no
Through th
H/D exchange
tein folding. Th
disulfide bond
ride or urea in
peptide nitrog
ing is then initi
the denaturant
neously lower
neutrality, hyd
OH$ and, ther
After a preset
(using a third
called labeling
tide nitrogen a
drogen bonds
that are hydro
hydrogen exch
(10 to 40 ms),
lowering the p
lowed to go to
changeable site
2D proton NM
signed). By rep
at position 93; the other is the site at which an O2 molecule binds. Within this pocket, the accessibility of the
heme group to solvent is highly restricted. This is important for function, because free heme groups in an oxygenated solution are rapidly oxidized from the ferrous
(Fe2!) form, which is active in the reversible binding of
O2, to the ferric (Fe3!) form, which does not bind O2.
Knowledge of the structure of myoglobin allowed
researchers for the first time to understand in detail the
Page 48
eAMtwo
3.4).
The lowest-priority substituent, often

hydrogen, is pointed away from the
viewer. The configuration about the
carbon is called S, from the Latin sinister for left, if the progression from
the highest to the lowest priority is
7552dc03_41-76 4/17/01 7:22 AM Page 43
Arginine
Lysine
counterclockwise.
The configuration
is
(Arg, R)
(Lys, K)
e and Function
called R, from the Latin rectus for
right, if the progression is clockwise.
1: Positively Charged R Groups

"
Histidine
(His, H)
NH3+
H2C
C
CH
C
CH2
N43
!N
CH
C
C CH3
Fe
CH
CH
(a)
(b)
CH2
Notation for distinguishing stereoisomers
O2
FIGURE 417 The

heme group. This group is present in myoglobin,
The four different substituents
of an
hemoglobin, cytochromes, and many other heme proteins. (a) Heme
asymmetric carbon atom are assigned
consists of a complex organic ring structure, protoporphyrin, to which
a priority according istobound
atomic
number.
an iron atom in its ferrous (Fe2!) state. The iron atom has six
The lowest-priority substituent,
often
coordination bonds,
four in the plane of, and bonded to, the flat porhydrogen, is pointed
away
from and
thetwo perpendicular to it. (b) In myoglobin and
phyrin
molecule
hemoglobin,
one the
of the perpendicular coordination bonds is bound
viewer. The configuration
about
to a nitrogen
atom of
a His residue. The other is open and serves as
carbon is called S, from
the Latin
sinisthe binding site for an O molecule.
ter for left, if the progression from 2
the highest to the lowest priority is
counterclockwise. The configuration is
called R, from the Latin rectus for
right, if the progression is clockwise.
H
N
H2C
(3)
C H
+H N
3
CH2
NH3+
and
COO
+
COO
+
FIGURE 3.4 The
NHL3and
The
CH2
H3N
isomer
COO
H3N
pKa=4.0
isomer
NH2
H2N
R refers to the side chain.
isomers of amino acids.
C
isomers are mirror images of each other.
NH
CH2
7552dc03_41-76
AAs are Zwitterions! (Why?)

N
H C
H+
H
H+
C
N
CH
H
H2N
Imidazole
R
COO
7:22 AM
CH2
C
C
O
ization.
protons near
+H N
Group3
NH2
1: Properties of AA: pKa

CH2
COO
+H N
3
Acid
O
COO
Typical pK *
Base
3.1
+H N
3
COO
Why are pKas important?

H
2
18
CH2
CH2
CH2
Terminal -carboxyl group
CH2
+H N
3
COO
Asparagine
Note.
in biological systems, theirGlutamine
pKas can
(Gln, Q)
(Asn, N)
O
Aspartic acid
be
substantially
shifted from its intrinsic
4.1
C
C
H
Glutamic acid
O
O
pKa to
apparent
pKa due to the
FIGURE 3.16 Amino Hacids with side-chain carboxylates
and
carboxamides.
interactions
when
buried in a biomolecular
N
N
6.0
+
Histidine
environment.
N
N
Aspartate
(Asp, D)
(from the Latin sinister meaning left).

The counterclockwise direction of the
arrow from highest- to lowest-priority
substituents indicates that the chiral
center is of the S configuration.
O
Glutamate
(Glu,
O E)
Terminal -amino group
pH = log "# H + $%
+ H
H
H
8.0
H
H
glutamateasparagine
and glutamineeach of which contains a terminal
H
Cysteine
8.3
S
S
carboxamide in place
of a carboxylic
acid (Figure 3.16).
H
O
O
Tyrosine
Seven of the 20 amino acids have 10.9
readily ionizable side chains. These
7 amino acids are able
to donate or accept protons to facilitate reactions
H
N
N
Lysine
10.8
H
H
as well as to form ionic
bonds. Table
3.1 gives equilibria and typical pKa
H
H
H of the sideH chains of tyrosine, cysteine, arginine, lyvalues for ionization
N H
N
H
sine, histidine, Hand
glutamic
acids in proteins. Two other
Arginine
12.5
N C aspartic Nand
C
N H
N H
groups in proteinsthe
terminal
!-amino
group and the terminal !H
H
carboxyl groupcan be ionized, and typical pKa values are also included
pK values depend on temperature, ionic strength, and the microenvironment of the
ionizable
group.
in Table
3.1.
Amino
acids are often designated by either a three-letter abbreviation or
a one-letter symbol (Table 3.2). The abbreviations for amino acids are the
first three letters of their names, except for asparagine (Asn), glutamine
"# A $%
10
[ HA ]
amino acids is altered by a change in pH. If the pH is 2 units below the pKa, how much will be protonated?
The zwitterionic form predominates near if the pH is equal to the pKa, how much will be protonated?
If the pH is 2 units above the pKa, how much will be protonated?
physiological pH.
TABLE 3.1 Typical pKa values of ionizable groups in proteins
"#A $%!"H #$
Ka =
[ AH ]
FIGURE 3.6 Ionization state as a

function of pH. The ionization state of
NH2
CH2
50
With a pKa value near 6, the imidazole group can be uncharged or positively charged near neutral pH, depending on its local environment (Figure
3.15). Indeed, histidineZwitterionic
is often found
in the active sites
of enzymes, where
form
Both groups
a
deprotonated
the imidazole ring can bind and release protons in the
course of enzymatic
reactions.
The set of aminoBoth
acids
also contains two with acidic side chains: aspargroups
tic acid and glutamicprotonated
acid (Figure 3.16). These amino acids are often called
10
FIGURE 3.6 Ionization state as a
aspartate and glutamate to emphasize that their side chains are usually negfunction
of
pH.
The
ionization
state
of
atively charged at physiological pH. Nonetheless, in some proteins these
amino acids is altered by a change in pH.
side0 chains 2do accept
and this
ability
impor4 protons,
6
8
10 is often
12 functionally
14
The zwitterionic form predominates near
pH. = pK
tant. In addition, the set includes
and pH
pH uncharged derivatives of aspartate physiological
+ log
Concentration
Page 50
!!
!
A + H +
HA
!
H2N
The side chains are usually negatively

charged at physiological CpH.
O
O
O C
H2C
H2C
(Why?)
CH2
CH2
CH2
CH2
H sites of enzymes
H chains maybe present
H
H and
at the active
These side
+
+H metal
+H N
maybe+Hinvolved
inbinding
ions.
H3N
COO
COO
COO
COO
3N
3N
3
positively charged residues. O
They can form salt bridgesO with

O
NH
pK = log10 K a
CH
pKa=4.5
O
O C
acids have an S absolute configuration
4/17/01
17
1: Properties of AA: pKa
Salt bridge
COO
Only L amino
acids are constituents of proteins.
For almost all amino
N acids,
CH2
CH
+2
COO
CH
NH3has
the L isomer
S (rather than R) absolute configuration (Figure
HC 3.5). AlCH
CH
(3)
R
2
2
though
considerable
effort has
acids
in
UOW
CHEM320
2016, Haibo
Yu gone into understanding why amino
C
N
proteins haveCHthis absolute configuration,
CH2
CH2no satisfactory explanation has
2
H (4)
been arrived at. It seems plausible that the selection of L over D was arbiH3N inCevolutionary
COO
C made,
COO was fixed+early
trary+H
but,
history. +H3N C COO
3N once
Amino acids in solution at neutral pH exist predominantly as dipolar
(2)
C
(1)
H
H
H
ions (also called zwitterions). In the dipolar form, the amino group is proton+
#
"
Histidine
Lysine
COO
NH3
ated
(NH(Lys,
) K)
andOnly
the carboxyl
groupArginine
is
deprotonated
(COO
).
The
ionFIGURE
L amino
acids
are
3 3.5
(His, H)
(Arg, R)
ization
an amino acid
varies with
(Figure 3.6). In acid solution
foundstate
inofproteins.
Almost
all pH
L amino
(e.g., pH 1), the amino group is protonated (NH3") and the carboxyl group
have an(COOH).
S absolute
configuration
isacids
not dissociated
As the
pH is raised, the carboxylic acid is the
first
group
give upsinister
a proton,meaning
inasmuch asleft).
its pKa is near 2. The dipolar
(from
theto Latin
FIGURE 3.5 Only L amino acids are
form persists until the pH approaches 9, when the protonated amino group
found in proteins. Almost all L amino
Glutamine
(Gln, Q)
Tertiary Structure of Small Globular Proteins, III. Lysozyme
NH3+ +
COO
Asparagine
(Asn, N)
Glutamate
(Glu, E)
Ribonuclease, another small globular protein (Mr

13,700), is an enzyme secreted by the pancreas into the
small intestine, where it catalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested
(basic amino acids) (Why?);

H (4)
with both protonated and neutral forms at
His is equilibrium
physiological pH. His may be present at the active site of enzymes
(2)
(proton
donor/acceptor)
and involved in binding metal ions.
C
(1)
H
N
N
H
CH2
Guanidinium
H+
The counterclockwise direction of the

NH2
+
arrow from highest- to lowest-priority
C
R chiral
R
H+
substituents
indicates
that
H
H
NH2 the
H2N
center
is
of
the
S
configuration.
+H N
+
COO
COOH
H3N
3
C CH3
C
Fe
pKa=6.5
CH2
H2C
C
N
N!
CH2
Lys and Arg are usually protonated (charged) at physiological pH (?)
polar
otonionution
roup
is the
ino acids
polar
ine.
roup
CH3
NH2
CH
C Acids
C
A Repertoire CH
of 20C Amino
HN
C
CH2
CH2
CH3 C
pKa=12
pKa=10.5
Amino acids are the building blocks of proteins. An !-amino acid consists
of a central carbon atom, called the ! carbon, linked to an amino group, a
carboxylic acid group, a hydrogen atom, and a distinctive R group. The R
group is often referred to as the side chain. With four different groups connected to the tetrahedral !-carbon atom, !-amino acids are chiral; the two
mirror-image forms are called the L isomer and the D isomer (Figure 3.4).
H2N
1: Negatively Charged R Groups
"
CH2
CH2
3.1 PROTEINS ARE BUILT FROM A REPERTOIRE

OF 20 AMINO ACIDS
acids,
. Alds in
n has
arbi-
in which a polypeptide chain can be folded. In Figure

418 the structures of cytochrome c, lysozyme, and
ribonuclease are compared. These proteins have different amino acid sequences and different tertiary structures, reflecting differences in function. All are relatively
small and easy to work with, facilitating structural analysis. Cytochrome c is a component of the respiratory
chain of mitochondria (Chapter 19). Like myoglobin, cytochrome c is a heme protein. It contains a single
polypeptide chain of about 100 residues (Mr 12,400) and
a single heme group. In this case, the protoporphyrin of
the heme group is covalently attached to the polypeptide. Only about 40% of the polypeptide is in !-helical
segments, compared with 70% of the myoglobin
chain.
Aspartate
The rest of the cytochrome c chain contains # turns and
(Asp, D)
irregularly coiled and extended segments.
Lysozyme (Mr 14,600) is an enzyme abundant in egg
white and human tears that catalyzes the hydrolytic
cleavage of polysaccharides in the protective cell walls
of some families of bacteria. Lysozyme, because it can
lyse, or degrade, bacterial cell walls, serves as a bactericidal agent. As in cytochrome c, about 40% of its 129
amino acid residues are in !-helical segments, but the
arrangement is different and some #-sheet structure is
also present (Fig. 418). Four disulfide bonds contribute stability to this structure. The ! helices line a
long crevice in the side of the molecule, called the active site, which is the site of substrate binding and catalysis. The bacterial polysaccharide that is the substrate
Protein Architecture
for lysozyme fits into this crevice.
19
20
1: Properties of AA: pI
units per milligram of total protein (Fig. 323). The spepK2 #

cific activity isGlutamate
a measure of enzyme
purity: it increases
10
9.67 becomes maximal
during purification
of an enzyme and
and constant when the enzyme is pure (Table 35).
8
pK1
pK #
1
Protein
2.19
pK2
!
3
(a)
Amino Acids Differ in Their

Acid-Base Properties
1: Hydrophobicity
Index
44
Methyl-substituted
carboxyl and
amino groups
Glycine
(Gly, G)
CHAPTER 3 Protein Structure and FunctionH
CH3
CH3
COOH
Alanine
(Ala, A)
H!
CH3 NH3
COO#
CH3 NH2
shared
properties ofof
many
aminomolecules
acids permit some
H
H
Hydrophobic Effect: based The
on
the
tendency
polar
to
Methylamine
Acetic acid
simplifying
generalizations
about
their
acid-base behavThe normal pK for an
The normal pK for a
7552dc03_41-76
4/17/01
7:22
AM
Page
44
exclude non-polar molecules
(oil
and
water
hate
each
amino group
is about
carboxyl
group
is about
4.8.
iors.
First,
all amino
acids
with
a10.6.
singleother).
!-amino group,
a
single
!-carboxyl
group,
and
an
R group that
does not
Hydrophobicity
Index: Hthe relative
hydrophobicity
among
amino
Carboxyl and
H
NH
NH
NH
amino groups
CH
ionize
have
titration
curves
resembling
that
of
glycine
H
H
H
H C COO
C COO
acids. in glycine H C COOH H (Fig.H310).
TheseH Namino
haveH NveryCOOsimilar, alH COO acids
FIGURE
3.7 Structures of glycine and
H
H
H
alanine.!-Amino
(Top) Ball-and-stick
models
!-Amino
acid
(glycine)
acid (glycine)
44
Glycine
Alanine
though
not identical,
pK
a values: pKa of the OCOOH
show the arrangement
of atoms and
pK "
pK " 2.34
(Gly, G)
(Ala,
A) 9.60
!
Question: Which group

of amino acids are more
hydrophobic?
Electronegative oxygen atoms

H
in the carboxyl group pull electrons
away
the
+Hfrom
C amino
COOgroup,
3N
lowering its pKa.
H
pKa of the R
3N
COO
H
Alanine
(Ala, A)
Glycine
(Gly, G)
perturbations of pKa are due to intramolecular interactions. Similar efcaused by chemical groups that happen to be positioned
example, in the active site
CH3 of an enzyme.
H
HValine
H
Leucine
Isoleucine
values for the ionizableGlutamate

groups in glycine are lower
than those for sim-Glutaminefects can be
Asparagine
Aspartate
(Asn, N)
(Asp,
E) and carboxyl groups.
ple, D)methyl-substituted (Glu,
amino
These downward(Gln, Q) nearbyfor

arrangement of bonds around atoms (see
O
C O
O
representation
(see Chapter 1 Appendix).
C
H
COO
+H N
3
COO
CH2
+H N
3
(Val, V)
+H N
3
C
CH2
H
+
COO
H3N
+H N
3
H
Alanine
(Ala, A)
NH2
H3C
NH2
CH2CH
CH3
Isoleucine
(Ile, I)
H
COO
+
3N
+HHN
3
COO
COO
+H N
3
LeucineO
(Leu, L)
CH2
COO
H3N
CH2
COO
CH2
C
COO
COO
CH3
HC
H
+H
3N
Methionine
(Met, M)
H2C
CH2
COO
H
+H
3N
H2C
CH3
*
C H
COO
+H
3N
Fraction number or volume of effluent
CH
Fractions sequentially collected
Figure 6-6 Ion exchange chromatography using stepwise

elution. Here the tan region of the column represents the ion
exchanger and the colored bands represent the various proteins.
(a) The protein mixture is bound to the topmost portion of the
ion exchanger in the chromatography column. (b) As the elution
progresses, the various proteins separate into discrete bands as a
consequence of their different affinities for the ion exchanger
under the prevailing solution conditions. Here the first band of

protein (red) has passed through the column and is being isolated
as a separate fraction, whereas the other, less mobile, bands
remain near the top of the column. (c) The salt concentration in
the elution buffer is increased to increase the mobility of and
thus elute the remaining bands. (d) The elution diagram of the
protein mixture from the column.
See the Animated Figures
Ion Exchange Chromatograph
positive and negative charges can bind to both cation and

Page 78 mac111 mac111:reb:
22
Abbreviation/
symbol
Nonpolar, aliphatic
R groups
Glycine
Gly G
Alanine
Ala A
Titration curves forProline
(a) glutamateProand
P
Valine
Val V
group is designated here
Leucine as pKR. Leu L
Isoleucine
Ile I
Methionine
Met M
Aromatic R groups
Phenylalanine
Phe F
Tyrosine
Tyr Y
Tryptophan
Trp W
Polar, uncharged
R groups
Serine
Ser S
Threonine
Thr T
Cysteine
Cys C
Asparagine
Asn N
Glutamine
Gln Q
Positively charged
R groups
Lysine
Lys K
Histidine
His H
Arginine
Arg R
Negatively charged
R groups
Aspartate
Asp D
Glutamate
Glu E
3.0
Mr
pK1
(OCOOH)
pK2
(ONH!
3)
pKR
(R group)
Hydropathy
index*
Occurrence in
proteins (%)
5.97
6.01
6.48
5.97
5.98
6.02
5.74
!0.4
1.8
1.6
4.2
3.8
4.5
1.9
7.2
7.8
5.2
6.6
9.1
5.3
2.3
pI
75
89
2.34
2.34
9.60
9.69
117
131
131
149
2.32
2.36
2.36
2.28
9.62
9.60
9.68
9.21
165
181
204
1.83
2.20
2.38
9.13
9.11
9.39
10.07
5.48
5.66
5.89
2.8
!1.3
!0.9
3.9
3.2
1.4
105
119
121
132
146
2.21
2.11
1.96
2.02
2.17
9.15
9.62
10.28
8.80
9.13
8.18
5.68
5.87
5.07
5.41
5.65
!0.8
!0.7
2.5
!3.5
!3.5
6.8
5.9
1.9
4.3
4.2
146
155
174
2.18
1.82
2.17
8.95
9.17
9.04
10.53
6.00
12.48
9.74
7.59
10.76
!3.9
!3.2
!4.5
5.9
2.3
5.1
133
147
1.88
2.19
9.60
9.67
3.65
4.25
2.77
3.22
!3.5
!3.5
5.3
6.3
(b)115
histidine.
1.99 The10.96
Average occurrence in more than 1,150 proteins. From Doolittle, R.F. (1989) Redundancies in protein sequences. In Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G.D., ed.), pp. 599623, Plenum Press, New York.
CH3
Chromatography
column
*A scale combining hydrophobicity and hydrophilicity of R groups; it can be used to measure the tendency of an amino acid to seek an aqueous
environment (! values) or a hydrophobic environment (" values). See Chapter 11. From Kyte, J. & Doolittle, R.F. (1982) A simple method for
displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105132.
H3C
CH3
High salt
cations. Polyanions and polycations therefore bind to anion

and cation exchangers, respectively. However, proteins and
other polyelectrolytes (polyionic polymers) that bear both
10:20 AM
2.0
Ave. Mw:
110 D
CH3
COO
H2N
Glycine
(Gly, CG) O
H2C
CH2
H
NH2
O Valine
UOW CHEM320
2016, HaiboCH
Yu2
C
O
Methionine
(Met, M)
(Ile, I)
COO
H3N
COO
H3N
CH2
+H N
3
(Leu, L)
(Val, V)
H3N
H2C
CH2
1.0
FIGURE 312
CH3
+H
Sample mixture
Low salt
1: Properties of Amino Acids
pK1 #
1.82
(b)
High-salt
elution
buffer
Buffer
pH=8
Buffer
pH=5.8
changer. A small volume of the impure protein solution is

applied to the top of a column in which the ion exchanger
has been packed, and the column is washed with this buffer
solution.
Various proteins bind to the ion exchanger with differanion exchangers depending on their net charge. The affinent affinities. As the column is washed with the buffer, a
ity with which a particular polyelectrolyte binds to a given
process known as elution, those proteins with relatively low
ion exchanger depends on the identities and concentrations
affinities for the ion exchanger move through the column
of the other ions in solution because of the competition
faster than the proteins that bind to the ion exchanger with
among these various ions for the binding sites on the ion exhigher affinities.This occurs because the progress of a given
changer. The binding affinities of polyelectrolytes bearing
protein through the column is retarded relative to that of
acidbase groups are also highly pH dependent because of
78
Chapter 3 Amino Acids, Peptides, and Proteins the variation of their net charges with pH. These principles
the solvent due to interactions between the protein molecules and the ion exchanger.
are used to great advantage in isolating biological moleThe greater the binding affinity of a protein for the ion
cules by ion exchange chromatography (Fig. 6-6), as deexchanger, the more it will be retarded. Thus, proteins
scribed below.
that bind tightly to the ion exchanger can be eluted by
In purifying a given protein (or some other polyelecTABLE 31 Properties and Conventions Associated
withthe
thepHCommon
AcidsofFound
in soProteins
changing the elution buffer to one with a higher salt controlyte),
and the saltAmino
concentration
the buffer
centration (and/or a different pH), a process called steplution in which the protein is dissolved are chosen so that
pKdesired
wise elution.
the
protein is strongly bound to the selected ion exa values
Amino acid
a
bondsRepulsion
in space. (Middle)
betweenStereochemically
the amino
realistic
formulas
show
the geometrical
group
and the
departing
proton
arrangement
of bonds
around
atoms (see
7552dc03_41-76 4/17/01 7:22 AM Page 49
lowers the
pKa for
the carboxyl
Chapter
1 Appendix).
(Bottom)
Fischer
group,
and oppositely
charged
groupsshow
lower
pKaas
bybeing
stabiprojections
allthe
bonds
lizing
zwitterion.
perpendicular
forthe
a simplified
FIGURE 311 Effect of the chemical environment on pKa. The pKa
(d)
(c)
OH" (equivalents)

pKR #
6.0 12/23/03
8885d_c03_078
(b)
Low-salt
elution
buffer
pK2 #
9.17
8
pH 6
Buffer
pH=5.8
Buffer
pH=8
Chapter 6. Techniques of Protein and Nucleic Acid Purification
10 Histidine
Page 136
"
2:25 PM
"
2/22/10
(a)
Pepsin
#1.0
Egg albumin
4.6
1.0
2.0
3.0
0
Serum
albumin "
4.9
OH (equivalents)
Urease
5.0
"-Lactoglobulin
5.2
Hemoglobin
6.8 COO
COO
COOH
COO
7.0
H N CH
H N CH
CH Myoglobin
H N CH
CH
CH
CH
CH Chymotrypsinogen
9.5
H
H
H
H
N
N
N
C
C cN
C
C
Cytochrome
10.7
CH pK
CH pK
CH pK
Lysozyme C N
11.0 C N
C
N
C
N
"
H3N
136
pI
JWCL281_c06_129-162.qxd
6
Isoelectric Points
TABLE
36pKRThe
#
of Some Proteins
4.25
4
pH
1: The Isoelectric Points of Proteins
Protein concentration
the titration curve of an amino acid is the relationship

tecting
and its
quantifying
that
protein
the
of
between
net electric
charge
andinthe
pHpresence
of the solumany
other
proteins
at point
each stage
of the between
procedure.
tion.
At pH
5.97, the
of inflection
the
Often, purification must proceed in the absence of any
8885d_c03_083 12/23/03 10:21 AM Page 83 mac111 mac111:reb:two stages in its titration curve, glycine is present preinformation
about
the
size and
physical
properties
of the
dominantly
as its
dipolar
form,
fully ionized
but with
no
protein
or about
the fraction
of the
total
protein mass
net electric
charge
(Fig. 310).
The
characteristic
pH
it represents
in the
extract.
For
proteins
that
are
at
the
net electric
charge
is called
the
The isoelectric point (pl)
ofwhich
a protein
is the
pHAcids
atis zero
which
its ennet
3.1 Amino
83 tissue extract
zymes,
the
amount
in
a
given
solution
or
isoelectric point or isoelectric pH, designated pI.
charge is zero.
canFor
be measured,
or assayed,
in terms group
of theincatalytic
glycine, which
has no ionizable
its side
From the titration curve of glycine we can derive
NH
NH
Neffect
H
the
enzyme
producesthat
is,gives
the
increase
in
chain,
the
isoelectric
is simply
arithmetic
mean
several
important
pieces point
of information.
First, itthe
a
CH
CH
CH
For
AA
w/o
sidechain:
quantitative
measure
of
the ionisable
pK of each
the two
iontheofrate
at
which
its
substrate
isofconverted
to reaction
the
two
pK
values:
a
COO
COO
COOH
izing groups: 2.34 for the OCOOH group and 9.60 for
productsthewhen
enzyme
is present.
For
ONH the
Note that the
group
of this purpose
13
1 group.
1carboxyl easily
glycine
100 times
more
acidic (more
ionpI
#is over
$$ (1)
(pK
! pK
(2.34 ! 9.60)
# 5.97
Glycine
one must
know
overall
1the
2) # $$equation
2 acid, which, asof the reaction
ized) than 2
the carboxyl group of acetic
pK " 9.60
we saw
in Chapter
2, has a pK ofprocedure
4.76about average
catalyzed,
(2)
an analytical
for determining
a carboxylin
group
attached
to an otherwise
unsubis for
evident
Figure
310,
glycine
hasappearance
a net negative
theAs
disappearance
the substrate
or pK
the
of
stituted aliphatic of
hydrocarbon.
The perturbed
of
charge
at
any
pH
above
its
pI
and
will
thus
move toward
glycine
is
caused
by
repulsion
between
the
departing
a reaction
product,
(3)
whether
the
enzyme
requires
coproton and the nearby positively charged amino group
7
the positive
(the
when (4)
placed
an
factors
such
aselectrode
metal
orinanode)
coenzymes,
theindeon the !-carbon
atom, asions
described
Figure 311. The
pH
pI " 5.97
opposite
charges
on
the resulting
zwitterion
are
stabielectric
field.
At
any
pHactivity
below
its
pI,
glycine has
a net
pendence
of
the
enzyme
on
substrate
concenlizing, nudging the equilibrium farther to the right. Simpositive
and
will
move
toward
elecilarly,
the
pK ofoptimum
the
amino
group
in glycine
is perturbed
pK " 2.34
tration,
(5)charge
the
pH,
and
(6)the
a negative
temperature
downward
relative to the average
pK of an amino
group.
trode
(the
cathode).
The
farther
the
pH
of
a
glycine
sozone in This
which
stable and
has high activeffect the
is dueenzyme
partly to the is
electronegative
oxygen
is from
its isoelectric
point,
the
atoms
in the carboxyl
groups, which tend
to pull
elec-greater the net
ity. lution
Enzymes
are
usually
assayed
at
their
optimum
pH
trons toward them, increasing the tendency of the amino
electric
charge
of the population
of glycine molecules.
group to convenient
give up a proton. Hence,
the !-amino group
0
and
at some
temperature
within the range
1
1.5
2
0.5
0
a pK for
thatexample,
is lower than that
of an aliphatic
At pHhas1.0,
glycine
existsamine
almost entirely as
OH (equivalents)
such as methylamine
(Fig. 311). In short, the pK of
!
the form
H3group
NOCH
any functional
is greatly
affected by itswith
chemicala net positive
2OCOOH,
FIGURE 310 Titration of an amino acid. Shown here is the titration
environment,
phenomenon
sometimes
exploited
in the
curve of 0.1 glycine at 25 $C. The ionic species predominatingcharge
at
of 1.0.aAt
pH 2.34,
where
there
is an equal mixactive sites of enzymes to promote exquisitely adapted
key points in the titration are shown above the graph. The shaded
!
!
depend on the
perturbed
ture reaction
of Hmechanisms
and
H3pK
NOCH2OCOO",
boxes, centered at about7552dc03_41-76
pK " 2.34 and pK 4/17/01
" 9.60, indicate
3NOCHthat
2OCOOH
7:22 the
AM re-Page 44
values of proton donor/acceptor groups of specific
gions of greatest buffering power.
the average
residues. or net positive charge is 0.5. The sign and
the
magnitude
of the net charge of any amino acid at 21
any
pH
can
be
predicted
in the 12
same way.
pK
2
4
6
8
10
CH2
COO
23
Amino Acids Can Be Classified by R Group

Knowledge of the chemical properties of the common
amino acids is central to an understanding of biochem-
24
listed in Table 31. Within each class there are gradations of polarity, size, and shape of the R groups.
Nonpolar, Aliphatic R Groups
The R groups in this class of
peptide bonds is an enzymatically controlled process that occurs on the ribosome and is directed
by the mRNA template. Although peptide bond formation can be reversed by the addition of
water (hydrolysis), amide bonds are very stable in water at neutral pH, and the hydrolysis of
peptide bonds in cells is also enzymatically controlled.
1: Peptides Are Chains of Amino Acids

R1
H
N
water
C
O
Hydrolysis
(protease)
water
amino terminus
(N terminus)
C
O
of proteins. Detailed consideration of the dynamic behav-
C
H
carboxyl terminus
(C terminus)
O
R2
amide bond: a chemical

bond until
formed
when a
is deferred
Chapter
carboxylic acid condenses with an amino group with
the expulsion of a water molecule.
backbone: the repeating portion of a polypeptide
chain, consisting of the NH group, the alpha-carbon
CH group, and the C=O of each amino-acid residue.
Residues are linked to each other by means of peptide
bonds.
separated charges that may be full or partial. Molecules

or functional groups having a dipole moment are said
to be polar.
phi torsion angle: see torsion angle.
polypeptide: a polymer of amino acids joined together

by peptide bonds.
hydrolysis: breaking a covalent bond by addition of a

molecule of water.
psi torsion angle: see torsion angle.
peptide bond: another name for amide bond, a

chemical bond formed when a carboxylic acid
condenses with an amino group with the expulsion of a
water molecule. The term peptide bond is used only
when both groups come from amino acids.
resonance: delocalization of bonding electrons over

more than one chemical bond in a molecule. Resonance
greatly increases the stability of a molecule. It can be
represented, conceptually, as if the properties of the
molecule were an average of several structures in which
the chemical bonds differ.
turns. However, before we begin our discussion of these baChapter 1 sic

From
Sequence
structural
motifs,toletStructure
us consider the geometrical properties of the peptide group because its understanding is
CUOW CHEM320
2016,NHaibo Yu
prerequisite to that of any structure containing it.
Examination of the geometry of the protein backbone re1902

Emilimportant
Fischer/Franz
Hofmeister
veals
several
features. First,
the peptide bond 25
is
essentially planar (Figure 3.23). Thus, for a pair of amino
A. The Peptide Group
C
acids linked by a peptide bond, six atoms lie in the same
C
In the 1930s and 1940s, Linus Pauling and Robert Corey plane: the !-carbon atom and CO group from the first
determined the X-ray structures of several amino acids
and dipeptides in an effort to elucidate the structural amino acid and the NH group and !-carbon atom from
constraints on the conformations of a polypeptide chain.
O
These studies indicated that the peptide group has a rigid, the second amino acid. The nature of the chemical bondplanar structure (Fig. 8-1), which, Pauling pointed out, is a ing within a peptide explains this geometric preference.
The peptide bond has considerable double-bond characO
ter, which prevents rotation about this bond.
2004 New Science Press Ltd
1: Peptide Bonds Have Double Bond

Character
R
RE 3.23 Peptide bonds are planar. In1.24

a pair of linked
acids, six atoms (C!, C, O, N, H, and C !) lie

in a plane.
123.5
argely determined
by as
its green balls. 120.5
Peptide bond
hains
are shown
C
e might naively suppose

sed of the same 20 types
uld be more or less alike
natured (unfolded) proeristics, a kind of homoandomly dangling side
mensional structure of a
protein is specified by its
a unique set of charac-
122
1.5
116
C
1.3
119.5
1.0
H
R
1.4
111
118.5
Amide
plane
C
O
H
N
1: A Peptide Bond Is Nearly Planar!
ically 1.32
plane, which is between the values expected for
a HCN single bond (1.49 ) and a CPN double bond
(1.27 ), as shown in Figure 3.24. Finally, the peptide
Figure 8-1 The trans-peptide group. The standard dimensions
bond
is uncharged,
allowing polymers of amino acids
(in angstroms, , and degrees, ) of this
planar group
were
derived by averaging the corresponding
quantities
the X-ray bonds to form tightly packed globular
linked
byinpeptide
crystal structures of amino acids and peptides. [After Marsh,
structures.
R.E. and Donohue, J., Adv. Protein Chem.
22, 249 (1967).]
See Kinemage Exercise 3-1
Two configurations are possible for a planar peptide
FIGURE 3.24 Typical bond lengths within a peptide unit.
Main unit
chain
bond. In the trans
configuration, the two !-carbon atoms
The peptide
is shown in the trans configuration.
221
are on opposite sides of the peptide bond. In the cis configuration, these groups are on the same side of the peptide bond. Almost
all peptide bonds in proteins are trans. This preference for trans over cis can
Side chainby the fact that steric clashes between groups attached to the
be explained
!-carbon
atoms hinder
formation
of theofcis form but do not occur in the trans
Figure 8-3 A polypeptide chain in its fully extended
conformation
showing
the planarity
configuration
3.25).
far the most common cis peptide bonds are
each of its peptide groups. [Illustration, Irving Geis.
Image from(Figure
the Irving
Geis By
Collection,
XPro
linkages. Such bonds show less preference for the trans configuration
Howard Hughes Medical Institute. Reprinted with
permission.]
because the nitrogen of proline is bonded to two tetrahedral carbon atoms,
limiting the steric differences between the trans and cis forms (Figure 3.26).
In contrast with the peptide bond, the bonds between the amino group
and the !-carbon atom and between the !-carbon atom and the carbonyl
26
group are pure single bonds. The two adjacent rigid peptide units may rotate
about these bonds, taking on various orientations. This freedom of rotation
chains. However, the three-dimensional structure of a

C
native (physiologically folded) protein is specified by its
primary structure, so that it has a unique set of charac1.24
teristics.
In this chapter, we shall discuss the structural features of
proteins, the forces that hold them together,Oand their hierarchical organization to form complex structures. This will
form the basis for understanding the structurefunction relationships necessary to comprehend the biochemical roles
R1
ior of proteins
and how they
fold to their native
structures very stable in water at neutral pH;
Peptide
Bond
(Amide
Bond):
9.
the lifetime ~ 1,000 years;
1 SECONDARY STRUCTURE
Directionality
is always N-terminus to C-terminus!
A polymers secondary structure (2 structure) is defined
as the local conformation of its backbone. For proteins, this 3.2.2 Polypeptide Chains Are Flexible
has come to mean the specification of regular polypeptide
Yet Conformationally
H
VMD
Prac:
Have a look at the peptide
bond! Restricted
backbone folding patterns: helices, pleated sheets, and
Definitions
ostable Proteins
res
CHAPTER
H
nd NMR Structures
dipole moment: an imaginary vector between two
osition
Synthesis
(ribosome)
-Dimensional
s of Proteins
-76
Figure 1-7 Peptide bond formation and

hydrolysis Formation (top to bottom) and
hydrolysis (bottom to top) of a peptide bond
requires, conceptually, loss and addition,
respectively, of a molecule of water. The actual
chemical synthesis and hydrolysis of peptide
bonds in the cell are enzymatically controlled
processes that in the case of synthesis nearly
always occurs on the ribosome and is directed
by an mRNA template. The end of a polypeptide
with the 7:22
free amino group
as the54
4/17/01
AM is known
Page
amino terminus (N terminus), that with the
free carboxyl group as the carboxyl terminus
(C terminus).
R2
Page 221
A. The Peptide Group
within
a peptide
explains
geometric preference.
bond,
such
as a CC
bond,this
is cylindrically
symmetrical
steric interference, which causes ing
In the 1930s and 1940s,
"1 Linus Pauling and Robert Corey
peptide
bond
about
itsamino
bond
axis,has
so considerable
that we mightdouble-bond
expect such acharacbond to
less The
the cis conformation (Fig. 8-2) to determined
be !8 kJthe
! mol
X-ray structures
of
several
acids
5 Quaternary Structure
and dipeptides
in an effort to ter,
elucidate
the
structural
which
prevents
rotation
thiscase,
bond.
exhibit
free
rotation.
If this about
were the
then in ethane,
stable than the trans conformation (this
energy difference
A. Subunit Interactions
constraints
on the
of
a polypeptide
chain.
B. Symmetry in
for
example, all
torsion angles about the CC bond would
isProteins
somewhat
less inbonds
peptide
bonds
byconformations
a Pro
FIGURE
3.23 Peptide
are planar.
Infollowed
a pair of linked
These studies indicated that the peptide group has a rigid,
C. Determination of Subunit Composition
amino
acids,
sixin
atoms
N, the
H, and
C !residues
) liestructure
in a plane.
be equally
in ethane are
residue
and,
fact,(C!10%
Pro
in(Fig.
proteins
!, C, O, of
planar
8-1), which, Pauling
pointed likely.
out, isHa Yet certain conformations
H
Appendix: Viewing Stereo Pictures
Side
chains
arepeptide
shown asbond,
green whereas
balls.
favored Cdue to Nquantum mechanical
effects
arising from
follow
a cis
cis peptides are otherC
N+
the interactions
ofCits molecular orbitals.
The
wise extremely rare).
C
C staggered
C
O
conformation
180) is ethanes
O R(Fig. 8-5a; torsion angleO(
most stable
whereas
the eclipsed conformaa. Polypeptide Backbone Conformations May Be 1.24
Peptide bond resonance
structures
H arrangement,
tion (Fig. 8-5b; torsion angle ( 0) is least stable. The enDescribed by Their Torsion Angles
123.5
120.5
ergy
difference
between
staggered
and eclipsed
conabove
considerations
they
The properties of aThe
protein
are largely
determined byare
its important because
Peptide
bond
The
inability
of the
bondthe
to rotate
constrains
the conH
C
"1
C
three-dimensional
structure.
One
might
naively
suppose
, a quantity
that
formations
!12 kJ ! mol
indicate that the backbone of a protein is a51linked se- formation
122
of in
theethane
peptideis backbone
and accounts
for the
that since proteins are all composed of the same 20 types
1.
1
1.0groups
quence of rigid planar peptide

(Fig. 8-3). We
can
represents
an energy
barrier to free
rotationis about
the
116 .33
.46 111
of amino acid residues, they would be more or less alike
bonds
This double-bond
character
also ex1planarity.
N
CC
single bond. Substituents other than hydrogen
therefore
polypeptides
conformain their properties.
Indeed, specify
denatureda(unfolded)
pro- 1 backbone
C
118.5
in
the
length
of
the
bond
between
the
CO
and
119.5 pressed
.4
N
5
teins have rather

similar
characteristics,
a kind2 of(rotation
homo1.5
tion
by the
angles
or dihedral anexhibit greater steric interference; that is, they increase
1 angles
C torsion
NH
groups. The CN distance in a peptide bond is typ1.3
geneous average of their
randomly
dangling
side
Amide
of this energy barrier due to their greater bulk.
bond (%)
and the
gles) about the
C!N
C C!C bond (&) 1.0 the size
E. Protein Denaturation
This is partly a result of
F. Explaining the Stability of Thermostable Proteins
H
N+
1930s and 1940s Linus Pauling & Robert Corey
1: The Trans is Strongly Favoured!

=0
=180
FIGURE 3.25 Trans and cis peptide
7552dc03_41-76
7:22
Page 55favored
bonds.4/17/01
The trans
formAMis strongly
because of steric clashes that occur in the

cis form.
~50:1 trans:cis ratio for most AA pairs
O
Peptide bond resonance structures
The inability of the bond to rotate constrains the conformation of the peptide backbone and accounts for the
the structural features of
m together, and their hierdouble-bond character is also ex-CH2-NH- bonds planarity.
1.45ThisAmine
mplex structures. This will
pressed
in
the
length
of
the bond between the CO and
he structurefunction re
1.5
-CO-NH- NH groups.
1.32
Amide
end the
1 roles .32 See Kinemage Exercise 3-1
Cbiochemical
The CN distance in a peptide bond is typ1
C
-CH=N- 221ically 1.32
1.25
, whichImine
is between the values expected for
C
a CN single bond (1.49 ) and a CPN double bond
Resonance
- the delocalisation of electrons
several
atoms;
increase
polarity
the
1.24
(1.27over
), as
shown
in Figure
3.24.the
Finally,
theofpeptide
peptide bond in a way that carbonyl oxygen
has
negative
charge
while
amide
nitrogen
has
bond is uncharged, allowing polymers of amino acids
O
positive
charge.
linked by peptide bonds to form tightly packed globular
structures.
Two configurations are possible for a planar peptide
UOW
CHEM320
2016, Haibo
Yu a peptide unit.
27
RE 3.24 Typical
bond
lengths
within
bond. In the trans configuration, the two !-carbon atoms
eptide unit is shown in the trans configuration.
are on opposite sides of the peptide bond. In the cis con-
Cis
Trans
=180
55
Primary Structure
=0
Figure 8-1 The trans-peptide group. The standard dimensions

(in angstroms,
1.0,and degrees, ) of this planar group were
derived by averaging the corresponding quantities in the X-ray
crystal structures of amino acids and peptides. [After Marsh,
1.4J., Adv. Protein Chem. 22, 249 (1967).]
R.E. andN
Donohue,
5
FIGURE 3.26 Trans and cis XPro
Trans
Cis
bonds. The energies of these forms are

relatively balanced because steric clashes
occur in both forms.
~4:1 trans:cis ratio for X-Pro pairs;
about two bonds of each amino acid allows proteins to fold in many different
Isomerisation
0.1-1/sec
(~20
energy
barrier);
ways.
The rotationsrate
about
these bonds
cankcal/mol
be specified
by dihedral
angles
(Figure
The angleinofprotein
rotationfolding;
about the bond between the nitrogen
Rate 3.27).
determining
and the !-carbon atoms is called phi ("). The angle of rotation about the
Catalysed by an enzyme: Proline isomerase;
bond between the !-carbon and the carbonyl carbon atoms is called psi (#).
A clockwise rotation about either bond as viewed from the front of the back
UOW
CHEM320
2016, Haibo
group
corresponds
to aYupositive value. The ! and " angles determine the
path of the polypeptide chain.
Are all combinations of ! and " possible? G. N. Ramachandran recognized that many combinations are forbidden because of steric collisions be-
Dihedral angle
A measure of the rotation about a
bond, usually taken to lie between
#180 and $180. Dihedral angles are
sometimes called torsion angles.
28
a unique conformation. The favorable entropy associated with the large

number of conformations in the unfolded form opposes folding and must
be overcome by interactions favoring the folded form. Thus, highly flexible polymers with a large number of possible conformations do not fold into
Section 8-1. Secondary Structure 223
unique structures. The rigidity of the peptide unit and the restricted set of allowed " and # angles limits the number of structures
accessible
to theHunfolded
H
H
form sufficiently to allow protein folding to Hoccur. H
7552dc03_41-76
1: Rotations around &

H
(B)
(A)
N
H
H
C
R
C
O
H
N
O
C
H R
N
H
(a) Staggered
H
H
(C)
(b)
Eclipsed
(a)
(b)
Indicated
thesingle
Ramachandran
Diagram
acid in a polypeptide can be adjusted by rotation
about by
two
bonds. (A)
Phi (!) is the
The sterically allowed values of ! and " can be deterangle of rotation about the bond between the
nitrogen
and
the
%-carbon
atoms,
mined by calculating the distances between whereas
the atoms of a
psi (") is the angle of rotation about the bond
between
the
%-carbon
and
thethe
carbonyl
tripeptide
at all
values
of ! and
" for
central peptide
unit. Sterically
forbidden
as that
carbon atoms. (B) A view down the bond between
the nitrogen
and conformations,
the %-carbon such
atoms,
shown
Fig. 8-6,
are thosethe
in which
any nonbonding
showing how ! is measured. (C) A view down
the inbond
between
%-carbon
and the interatomic distance is less than its corresponding van der Waals
carbonyl carbon atoms, showing how " is measured.
distance. Such information is summarized in a conforma-
torsion angles/rotational angles/

dihedral angles :
C-N-C-C
map
or Ramachandran diagram (Fig. 8-7), which was
: tion
N-C-C-N
invented by G.N. Ramachandran.
1: Ramachandran Plot
Page 119 mac76 mac76:385_reb:
Figure 8-7 indicates that 77% of the Ramachandran diagram (most combinations of ! and ") is conformationally
inaccessible to a polypeptide chain. The particular regions
of the Ramachandran diagram that represent allowed conformations depend on the van der Waals radii chosen to
calculate it. But with any realistic set of values, such as that
in Table 8-1, only three small regions of the conformational
map are physically accessible to a polypeptide chain. Nevertheless, as we shall see, all of the common types of regular
secondary structures found in proteins fall within allowed
regions of the Ramachandran diagram. Indeed, the
H
N
C
H H
N
H
Try-Gly-Gly-Phe-Leu
CHAPTERpentapeptide
3 Protein Structure
and Function(YGGFL)4.1 Overview of Protein Structure+180119
shows the sequence from the amino
terminus to
the carboxyl terminus.
This
120
The carbonyl oxygen has a partial negative
O!"
O
O"
charge and the amide nitrogen a partial positive
pentapeptide, Leu-enkephalin, is an opioid
C
C !# C$
C # C$
charge, setting up a small electric dipole.
C$
peptide
that
modulates
the
perception
of
60
Virtually all peptide bonds in proteins occur in
N
C$
C$
N
C$
N
Tyr in
Gly
this trans configuration; an exception is noted
pain. The reverse
pentapeptide,
H
H Leu-PheH
Figure 48b.
0
Amino
Gly-Gly-Tyr (LFGGY), is a different molecule
FIGURE and
3.28shows
A Ramachandran
terminal
no such effects.
residue
diagram
showing the values of ! and
60
O
". Not all # and $R values are possible
Carboxyl
1.24
terminus
without collisions
between atoms. The
120
1.46
1.53
Ca
C
w
R1
most favorable
regions
shownf in dark
FIGURE
3.20 are
Components
of a
O
H
N
H
f
w
f
w
Ca green;
borderline
regions
are
shown
in
180
1.32
polypeptide chain. A polypeptide chain
C 120N 60
C0
180
light
green.
The
structure
on
the
right
is
consists
of
a
constant
backbone
(shown
in
Amino
C
C
N
terminus
H
disfavored
because
steric clashes.
H
black)
and of
variable
side chains (shown in
O
H
R2
green).
NCa
CaC
H H
C
O H2C
H
N
O H2C
Gly
C
H
N
H
Phe
CH3
CH3
H
C
Leu
Carboxyl
terminal residue
R3
H
N
60 C 120 +180
N
H
CN
FIGURE 42 The planar peptide group. (a) Each peptide bond has
some double-bond character due to resonance and cannot rotate.
(b) Three bonds separate sequential ! carbons in a polypeptide
chain. The NOC! and C!OC bonds can rotate, with bond angles
O
designated " and #, respectively. The peptide CON bond is not free
to rotate. Other single bonds in the backbone may also be
rotationally hindered, depending on the size and charge of the R
C
Ca
groups. In the conformation
shown, " and # are 180% (or " 180%).
Dalton
As one looks out from the ! carbon, the # and " angles increase as
A (respectively)
unit of mass
very nearly equal to that
N
the carbonyl or amide nitrogens
rotate clockwise.
(c) By convention, both " andof
# are
as 0% when
the twoNamed after John
a defined
hydrogen
atom.
H
peptide bonds flanking that ! carbon are in the same plane and
Dalton (17661844), who developed
positioned as shown. In a protein, this conformation is prohibited
the atomic
of matter.
by steric overlap between an !-carbonyl
oxygentheory
and an !-amino
hydrogen atom. To illustrate the bonds between atoms, the balls
representing each atom are smaller than the van der Waals radii for
this scale. 1 & 0.1 nm.
HC
R5
O
H
(C= 90, C
= 90)
C
N
C
Disfavored
H
H
O
R4
Ca
good
hydrogen-bond donor. These groups interact with each other and with
3.3 SECONDARY
STRUCTURE:
POLYPEPTIDE
CHAINS
functional groups
from side chains
to stabilize particular
structures, as will
N
CAN FOLD
INTOinREGULAR
STRUCTURES SUCH AS
be
discussed
detail.
O
THE ALPHA
HELIX,
THE BETAchains
SHEET,
ANDbetween
TURNS50 and 2000 amino
C
Most
natural polypeptide
contain
w
residues
and are commonly referred to as proteins. Peptides made of
AND acid
LOOPS
29
Ca
small
numbers of amino acids are called oligopeptides or simply peptides. The
H
f
Can a polypeptide
chain fold
intoofa an
regularly
structure?
In110,
1951,and so the
mean
molecular
weight
amino repeating
acid residue
is about
R
Linus Pauling
and
Robert of
Corey
twobetween
periodic 5500
structures
called We can
molecular
weights
mostproposed
proteins are
and 220,000.
the ! helix
theof"apleated
sheet
(betaispleated
sheet).
Subse-of daltons;
also(alpha
refer helix)
to the and
mass
protein,
which
expressed
in units
quently,one
other
structures
such
theatomic
" turn mass
and omega
loop were
idendalton
is equal
toasone
unit. (#)
A protein
with
a molecular
(c)
tified. Although
periodic,
common
turndaltons,
or loop or
structures
are well30
weight ofnot
50,000
has these
a mass
of 50,000
50 kd (kilodaltons).
defined andIn
contribute
with !the
helices
and
" sheets tochain
form the
final proteinThe most
some proteins,
linear
polypeptide
is cross-linked.
Kilodalton (kd)
FIGURE 43 Ramachandran plot for -Ala residues. The
common cross-links are disulfide bonds, formed by the oxidation of a pair of
conformations of peptides are A
defined
the mass
values ofequal
" and #. to 1000 daltons. structure.
unitbyof
Conformations deemed possible are those that involve little or no
#180
cysteine residues (Figure 3.21). The resulting unit of linked cysteines is
steric interference, based on calculations using known van der
L
1: Ramachandran Plot from PDB
Exercise 3-1
2:13 PM
FIGURE 3.19 Amino acid sequences

56 have direction. This illustration of the
b. Allowed Conformations
of Polypeptides
Are
FIGURE 3.27 Rotation about bonds in a polypeptide.
The structure
of each amino
C#N bond (!) and the C#C bond ("). The torsion angles are
both 180 in the conformation shown and increase, as is indicated,
in a clockwise manner when viewed from C#. [Illustration, Irving
Geis. Image from the Irving Geis Collection, Howard Hughes
Medical Institute. Reprinted with permission.]
See Kinemage
12/30/03
H3N
Figure 8-4 The torsional degrees of freedom in a peptide unit.
Page 56
Indeed, with large substituents, some conformations may

= 80
= +85
be sterically forbidden.
The 2016,
only reasonably
UOW CHEM320
Haibo Yufree movements are rotations about the
8:19 PM
H2C
8885d_c04_119
H
H
Figure 8-5 Conformations of ethane. Newman projections

indicating the (a) staggered
conformation and (b) eclipsed
conformation of ethane.
6/4/01
1: Disulfide Bond
STRUCTURAL INSIGHTS, appearing
Waals radii and bond angles. The areas shaded dark blue reflect
120
theoverlap
book,
molecular
conformationsthroughout
that involve no steric
andare
thus are
fully
allowed; medium
blue indicates conformations
allowed
at the
modeling-based
tutorials
that
enable you
60
extreme limits for unfavorable atomic contacts; the lightest blue
to review structure and learn what the
area reflects conformations that are permissible if a little flexibility is
0
research
tells ofustheabout
the
workings
allowed in thelatest
bond angles.
The asymmetry
plot results
from
the L stereochemistry
the amino acid residues.
The plots
other
of theof molecule.
To access,
goforto
the
"60
L-amino acid residues with unbranched side chains are nearly
Web site: www.whfreeman.com/biochem5,
identical. The allowed ranges for branched amino acid residues
"120
and
select
the chapter,
Structural
Insights,
such as Val, Ile,
and Thr
are somewhat
smaller than
for Ala. The Gly
residue, whichand
is lessthe
sterically
hindered, exhibits a much broader
title.
"180
"180
range of allowed conformations. The range for Pro residues is
greatly restricted because " is limited by the cyclic side chain to the
range of "35% to "85%.
w (degrees)
Statistical analyses of high resolution crystal structures
STRUCTURAL INSIGHTS, Elements of Protein Structure provides interactive

representations of some of the important elements of protein architecture described in
this chapter, including a Osummary of secondary structure motifs.
H
C
N
C
3.3.1 The Alpha Helix Is a Coiled StructureO Stabilized
H
H
C
N
H2C
by Intrachain Hydrogen
Bonds
C
S
H
In evaluating
potential
structures,
Pauling
and
Corey
considered
which conH2C
H
0
#180
This disulfide bond can join

separate polypeptide chains or
cross-link two Cysteines in
the
formations of peptides were sterically allowed and which most fully exS
ploited the hydrogen-bonding capacity of the backbone NH and CO
same chain to form Cystine;
+ 2H + 2e
Hovmller et al. 2002
f (degrees)
Cysteine
Oxidation
groups. The first of their proposed structures,

the !S helix, is a rodlike strucReduction
H coiled backbone forms the inner part of the rod
ture (Figure 3.29). A tightly
S
CH2
H
and the side chains extend outward in a helical array.
The ! helix is stabiCH2
C
lized by hydrogen bondsHbetween
the NH and CO groups
of the main chain.
N
C
C of each amino acid forms a hydrogen bond with
In particular, the CO group
H
N
O
the NH group of the H
amino Cacid that is
situated four residues
ahead in the
FIGURE 3.21 Cross-links.
The formation
Ribonuclease A
Chemical Reviews, 1998, Vol. 98, No. 3 1047
O except for amino acids near the ends of an !
of a disulfide bond from two cysteine sequence (Figure 3.30). Thus,
a bovine pancreas relies on the enzyme
maintaining
Cysteine
Cystine
residues is an oxidation reaction.
helix, all the main-chain itsCO
and and
NH
groups
aredrastic
hydrogen
bonded. Each
integrity
solubility
under
conditions:
0.25 N
5 C,
then,
pH helix
3.0
first, one
residue is related to the next
bysulfuric
a riseacid
of at1.5
and
along
the
axis and
at 95-100 C.84 The final step in this protocol calls
a rotation of 100 degrees,forwhich
gives of3.6
crystallization
theamino
enzyme. acid residues per turn of
Screw sense
The three-dimensional structure of RNase A is fully
helix. Thus, amino acidsencoded
spaced
three and four apart in the sequence are
by its amino acid sequence.85-89 This disDescribes the direction in which a helimade RNase
a favorite
model system
covery
spatially quite close to one
another
in anA !intohelix.
In contrast,
amino acids
cal structure rotates with respect to its
for the application of new methods to probe protein
two apart in the sequencefolding.
are situated
opposite
sides mass
of the
helix and so
axis. If, viewed down the axis of a heIn recent on
examples,
electrospray
spectrometry
haspitch
been used
to determine
disulfide
lix, the chain turns in a clockwise direcare unlikely to make contact.
The
of the
! helix,which
which
is equal to the
bonds (both native and nonnative) form during the
tion, it has a right-handed screw sense.
or a derivative
of the
reduced
molecule90-92
product of the translationfolding
(1.5 )
and
the number
of residues
per turn (3.6),
If the turning is counterclockwise, the
in which the eight cysteine residues are in mixed
is
5.4
.
The
screw
sense
of
a
helix
can
be
right-handed
or left92
Fourier (clockwise)
transform
with
glutathione.
disulfides
screw sense is left-handed.Figure 1. Ribbon diagram of the three-dimensional
infrared
spectroscopy, with
its uniquereveals
signa- that both
handed
(counterclockwise).
The(FTIR)
Ramachandran
diagram
The inscriptions
refer to the
structure of ribonuclease A.
Figure 8-6 Steric interference between adjacent residues. The

collision between a carbonyl oxygen and the following amide
hydrogen prevents the conformation ! $ %60, " $ 30.
[Illustration, Irving Geis. Image from the Irving Geis Collection,
Howard Hughes Medical Institute. Reprinted with permission.]
Bond Length ~2.05
See Kinemage Exercise 3-1.
VMD Prac: Identify 4 disulfide

bonds in Ribonuclease A.
The allowed region for Glycine is considerably larger (Why?)

The allowed region for Proline is much smaller (Why?)
72
location of the eight cysteine residues that give rise to the

four disulfide bonds, the two proline residues with cis
peptide bonds, and the three residues most important for
catalysis: His12, His119, and Lys41.
31
been used to characterize the structure

UOW CHEM320 2016, Haibo Yu copy has also6,69,70
of RNase B.
Altogether, over 70 sets of threedimensional coordinates related to RNase A have
been deposited in the Brookhaven Protein Data Bank
(www.pdb.bnl.gov).
RNase A is small. The mature enzyme, as secreted
ture for -sheets, has been used to probe new aspects

of RNase A folding.32,93-95 In these and other studies
on the folding of RNase A, the unfolded enzyme is
generated by high or low temperature, high or low
pH, or chaotropic agents. The unfolding of RNase A
by high pressure has attracted much interest, promising still more insights.96-101
Two distinct starting materials have been used in
most studies on the folding of RNase A: reduced
enzyme and oxidized enzyme (with the four native
disulfide bonds intact). Studies of the folding of the
32
such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as
chains.
We can calculate the approximate number of amino
acid residues in a simple protein containing no other
1: 2,3,4,5,6, ...
1: A Vast Range of Sizes in Biology
Dipeptides (2);
Tripeptides (3);
Tetrapeptides (4);
Pentapeptides (5);
... ...
Oligopeptides (a few);
Polypeptides (many, MW< 10,000);
TABLE 32
Cytochrome c (human)
Ribonuclease A (bovine pancreas)
Lysozyme (chicken egg white)
Myoglobin (equine heart)
Chymotrypsin (bovine pancreas)
Chymotrypsinogen (bovine)
Hemoglobin (human)
Serum albumin (human)
Hexokinase (yeast)
RNA polymerase (E. coli)
Apolipoprotein B (human)
Glutamine synthetase (E. coli)
Titin (human)
33
Summary on L1
1. 20 amnio acids and their properties (structure, pKa);
2. Important molecular forces (hydrophobicity/hydrophilicity,
hydrogen bonding, salt bridges);
3. Peptide bond ();
4. Ramachandran plot ( vs ).
Molecular Data on Some Proteins

Molecular
weight
Proteins;
For example, the amide bonds in the side chains of asparagine and glutamine are cleaved by acid treatment,
yielding aspartate and glutamate, respectively. The side
chain of tryptophan is almost completely degraded by
acid hydrolysis, and small amounts of serine, threonine,
35
13,000
13,700
13,930
16,890
21,600
22,000
64,500
68,500
102,000
450,000
513,000
619,000
2,993,000
Number of
residues
104
124
129
153
241
245
574
609
972
4,158
4,536
5,628
26,926
Number of
polypeptide chains
1
1
1
1
3
1
4
1
2
5
1
12
1
34

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Administration

UOW CHEM320 2016, Haibo Yu

UOW CHEM320 2016, Haibo Yu

L1-4: Hierarchy & Classification of Protein Structure

Molecular Modelling (Week 1-3)

Section 5-4. Gene Expression and Replication: An Overview

Central Dogma of Molecular Biology

What is the difference between CHEM320 and BIOL213/BIOL214?

We focus more on the physical forces underlying protein structure and

UOW CHEM320 2016, Haibo Yu

Figure 5-21 The central dogma of molecular biology. Solid

Proteins are the most versatile macromolecules in living systems and

Understand the hierarchy of protein structures

Protein engineering - de novo design novel enzymes for

UOW CHEM320 2016, Haibo Yu

Hierarchy of Protein Structures

UOW CHEM320 2016, Haibo Yu

1: What Is Primary Structure?

Primary the sequence of amino acids in a protein

UOW CHEM320 2016, Haibo Yu

The linear sequence of amino

UOW CHEM320 2016, Haibo Yu

of the set of 20 in that its side chain is bonded to both

1: Primary Structure - Amino Acids

1: Amino Acids Are Chiral Molecules

The four differen

viewer. The configuration about

Notation for disting

CHAPTER 3 Protein Structure and Function

1: The Genetic Codes Specify 20 Amino

FIGURE 3.7 Structures of glycine and

show the arrangement of atoms and

UOW CHEM320 2016, Haibo Yu

FIGURE 3.7 Structures of glycine and

show the arrangement of atoms and

1: AAs with Aliphatic Sidechains

centerH isC ofCHthe S configuration.

UOW CHEM320 2016, Haibo Yu

A Repertoire of 20 Amino Acids

FIGURE 3.10 Amino acids with

CHAPTER 3 Protein Structure and Function

UOW CHEM320 2016, Haibo Yu

Extinction coefficient (M1 cm1)

Since 1H has an NMR spectrum in a different frequency

absorbs light less strongly and at shorter wavelengths.

FIGURE 3.13 Structure of cysteine.

FIGURE 3.11 Absorption spectra of the aromatic amino acids

absorb strongly near 280 nm. [Courtesy of Greg Gatto].

FIGURE 3.12 Amino acids containing

threonine contain hydroxyl groups that

UOW CHEM320 2016, Haibo Yu

FIGURE 3.11 Absorption spectra of the aromatic amino acids

XH ' D2 O XD ' HOD

What is a Hydrogen Bond?

1: Polar and Uncharged R Groups

come together to form disulfide

in this spectral region (ranging into the tens of thousands;

Figure 9-10 UV absorbance spectra of the three aromatic

extinction coefficient (M-1 cm-1)

Extinction coefficient (M1 cm1)

where ! is the extinction coefficient [in units that are the

FIGURE 3.12 Amino acids containing

1: AAs with Aromatic Sidechains

What is Hydrophobic Interaction?