The Three

Dimensional
Structure of
Protein
(Chapter 6/7)
 Protein Structure
Many conformations are possible for proteins:
Due to flexibility of amino acids linked by peptide
bonds

At least one major conformations has

biological activity, and hence is considered
the proteins native conformation
Three-dimensional folding of the
protein myoglobin.
 The Four Levels of Organizations
in Proteins
1structure: the sequence of amino acids in a polypeptide chain

2structure: 3-dimensional arrangements (conformations) in

localized regions of a polypeptide chain; refers only to interactions
of the peptide backbone

3 structure: 3-D arrangement of all atoms

4 structure: arrangement of monomer subunits with respect to

each other
 1 Structure

The 1 sequence of proteins

determines its 3-D conformation
Changes in just one amino acid in
sequence can alter biological
function, e.g. hemoglobin associated
with sickle-cell anemia
 Primary Structure Determination

1. Determine which amino acids are present (amino

acid analysis)
2. Determine the N- and C- termini of the sequence
(a.a sequencing)
3. Determine the sequence of smaller peptide
fragments (most proteins > 100 a.a)
4. Some type of cleavage into smaller units necessary
Primary Structure Determination
 Amino Acid Composition

Total Hydrolysis followed by quantitative

analysis of the liberated amino acids

1. Acid Hydrolysis

2. Base Hydrolysis
N and C-terminal Determination
N-terminal Determination
 Peptide Sequencing
Can be accomplished by Edman Degradation
Relatively short sequences (30-40 amino
acids) can be determined quickly
So efficient, today N-/C-terminal residues
usually not done by enzymatic/chemical
cleavage
Peptide Sequencing
 Edman Degradation
Sequentially removes one residue at a time
from the amino end.
Phenyl isothiocyanate reacts with amino
group to form a phenylthiocarbamoyl
derivative.
Mild acid conditions create cyclic derivative.
Cyclic derivative is separated by
chromatography to identify amino acid
C-terminal Determination
 Protein Cleavage
Protein cleaved at specific sites by:
1) Enzymes- Trypsin, Chymotrypsin
2) Chemical reagents- Cyanogen bromide
Enzymes:
Trypsin- Cleaves @ C-terminal of (+) charged side
chains
Chymotrypsin- Cleaves @ C-terminal of aromatics
 Cleavage by CnBr

Cleaves @ C-terminal of INTERNAL methionines

Peptide Digestion
 Determining Protein Sequence
After cleavage, mixture of peptide fragments produced.
Can be separated by HPLC or other chromatographic
techniques
Use different cleavage reagents to help in 1 determination
SAMPLE PROBLEMS
State the cleavage pattern of the following
polypeptides by the indicated agents

S-A-F-K-P by chymotrypsin
T-C-G-M-N by CNBr
L-R-G-D by hydrazine
V-W-K-P-R-E by trypsin
An unknown decapeptide was isolated and
characterized. Complete hydrolysis of this
peptide gave : F(2), A,G,C,K,N,T, W and V.
Treatment with carboxypeptidase releases A.
Reaction with Edmans reagent gave PTH-T and a
nonapeptide. The nonapeptide was treated with
trypsin and gave 2 peptides: (V-C-G-A) and (N-F-
F-W-K). Give the sequence of amino acid in the
decapeptide.
You have isolated from a rare fungus an
octapeptide that prevents baldness and you wish to
determine its amino acid sequence. The amino
acid composition is K2, D, Y, F, G, S, A. Reaction of
the intact peptide with dansyl chloride yields
dansyl-A. Cleavage with trypsin yields peptides
whose compositions are: (K, A, S) and (G, F, K) plus
a dipeptide. Reaction with chymotrypsin releases
free D, a tetrapeptide with composition (k, S, F, A)
and a tripeptide whose composition following acid
hydrolysis is (G, K, Y). The enzymatic digests are
each carried out on the whole, undansylated
peptide.What is the sequence?
 Determine the sequence of the following
heptapeptide:
Amino acid analysis of the heptapeptide revealed that the original
peptide was composed of: R, V, Y, E, K, A and G.
Reaction of the heptapeptide with dansyl-Cl and acid hydrolysis
gave dansyl-A.
Digestion of the heptapeptide with:
carboxypeptidase gave G as the first detectable amino acid.
trypsin gave free R, a dipeptide (A-K) and a tetrapeptide
containing E, G, Y and V
digestion of the tetrapeptide above (derived from the trypsin
digestion above) with chymotrypsin gave two dipeptides: V-Y and
E-G.
pepsin gave a tetrapeptide and a tripeptide (Y-E-G).
Determine the amino acid sequence of a heptapeptide (2 M, D, R, K, F,
G) was isolated from the urine of a three-toed sloth, given the
following results:
Reaction of the heptapeptide with FDNB gave DNP-M.
Limited proteolysis with carboxypeptidase indicated that M was
the first amino acid released
Cyanogen bromide (CNBr) reaction with the heptapeptide
released one equivalent of free homoserine lactone
Chymotrypsin digestion of the heptapeptide yielded a
pentapeptide and a dipeptide. Reaction of the pentapeptide with
dansyl-Cl gave dansyl-M.
Trypsin digestion yielded two M-containing tripeptides and free
R.
Digestion of the heptapeptide with pepsin gave a tetrapeptide
(containing M, R, K and D) and a tripeptide (M, F, and G).
 The peptide glucagon from the Nile tilapia was treated with
chymotrypsin, and the resulting fragments were sequenced. A
second sample of the polypeptide was treated with trypsin, and the
fragments were sequenced. What is the sequence of the
polypeptide?

Chymotrypsin fragments Trypsin Fragments

LMNNKRSGAAE AQDFVR
SNDY WLMNNK
HSEGTF HSEGTFSNDYSK
LEDRKAQDF RSGAAE
VRW YLEDRK
SKY
 The sequence of crinia-angiotensin, an angiotensin II-like
undecapeptide from the skin of the Australian frog, is
determined. A single round of Edman degradation releases
DNP-Ala. A second round sample of the peptide is then treated
with chymotrypsin. Two fragments are released with the
following amino acid composition: fragment 1: (H,P,F,V) and
fragment 2 (A,D,R,G,P,I,Y). Next, a third sample of the
peptide was treated with trypsin, which results in two
fragments with the following amino acid compositions:
fragment III (A,D,R,G,P) and fragment IV ( H,I,P,F,Y,V).
Treatment of another sample with elastase yields three
fragments, two of which are sequenced: fragment V (H-P-F)
and fragment VI (A-P-G). What is the sequencre of the
undecapeptide?
 The amino acid sequence of a biologically active dodecapeptide was
determined by treating the dodecapeptide with chemical and enzymatic
reagents. The results obtained are given below:
Amino acid composition: F. G(2), I, K, L(2), Q, R, T, V, W
Hydrazine Treatment of the dodecaptide gave underivatized G
Trypsin digestion yielded 3 fragments:
Fragment A: F, K, L, T
Fragment B: G, V
Fragment C: G,I,L,Q,R,W (Edman reaction of this fragment
gave Pth-I
Chymotrypsin digestion yielded 3 fragments:
Fragment D: G(2), L,R,V (Edman reaction of this fragment gave
Pth-L)
Fragment E: F,T
Fragment F: I,K,L,Q,W (Edman reaction of this reaction gave
Pth-L.
What is amino acid sequence of the dodecapeptide?

37
Secondary Structure: Regular Ways
to Fold the Polypeptide Chain
The peptide bond has specific and
chemical constraint:
The peptide linkage can exist in either
CIS or TRANS configuration
 Almost all peptide
bonds in proteins are
TRANS
Rotation around the bonds in a
polypeptide backbone
 2 of proteins is hydrogen-bonded
arrangement of backbone of the
protein

Two bonds have free rotation:

1) Bond between -carbon and amino
nitrogen in residue
2) Bond between the -carbon and
carboxyl carbon of residue

44
A Ramachandran
plot of poly-L-
alanine
 Helices and sheets
Most common Secondary
Structures in Proteins
 -Helix
Coil of the helix is clockwise or right-
handed
Repeat every 18 residues equivalent to 5
turns
There are 3.6 amino acids per turn
Repeat distance is 5.4
Each peptide bond is trans and planar
C=O of each peptide bond is hydrogen
bonded to the N-H of the four amino acid
away
C=O----H-N hydrogen bonds are parallel
to helical axis
All R groups point outward from helix
The -Helix
Fig. 4-2a, p. 86
Model of Hemoglobin, the helical regions are
shown.

Fig. 4-2b, p. 86
 Several factors can disrupt an -helix

Proline creates a bend because of (1) the

restricted rotation due to its cyclic structure
and (2) its -amino group has no N-H for
hydrogen bonding
Strong electrostatic repulsion caused by the
proximity of several side chains of like
charge, e.g., Lys and Arg or Glu and Asp
Steric crowding caused by the proximity of
bulky side chains, e.g., Val, Ile, Thr
 -Pleated Sheet
Polypeptide chains lie adjacent to one another;
may be parallel or antiparallel
R groups alternate, first above and then below
plane
Each peptide bond is s-trans and planar
C=O and N-H groups of each peptide bond are
perpendicular to axis of the sheet
C=O---H-N hydrogen bonds are between
adjacent sheets and perpendicular to the
direction of the sheet
FIBROUS PROTEINS

Structural Materials of Cells

and Tissues
 Keratins

- and -keratins
 -Keratins
are the major proteins of hair and fingernails
and a major fraction of animal skin
are members of a intermediate filament group
contain long sequences-over 300 residues
Pairs of these right handed twist about one
another in the left handed coiled-coil structure
Arrangement of residues in a coiled coil.
This view down the axis of two seven-residue
shows that amino acids at 1 and 4 line up
on one side of each helix
 -Keratins
Contain more -sheets
Second major structural protein
Found mostly in birds and reptiles in
structures like feathers and scales
 Silk fibroin
The fibrous protein present in cocoons,
webs, nests and egg stalks
Consist of antiparallel sheets whose
chains extend parallel to the fiber axis
Contain six residue repeat
(-G-S-G-A-G-A)n

64
Silk fibroin sheets - sheets account for the
mechanical stability of silk

65
Theoretical model for the structure
of silk fibroin
 Collagen
Most abundant single protein in most
vertebrate
In large animal, may take up a third of
the total protein mass
The matrix material in bone
The major portion of tendons
Important constituent of the skin
 Tropocollagen
Basic unit of collagen fiber
A triple helix of three polypeptide chain
Each chain is 1000 residues long and left
handed with 3.3. residues/turn
These chains are wrap around each other
in a right handed sense
Rich in glycine and proline
Repetitive motif is G-X-Y
Molecular interactions
of collagen. Hydrogen
bonding in the collagen
triple helix. The
residues are stagerred
so that one G, X, Y
occur at every level
along the axis

69
Scurvy is caused by
Vitamin C deficiency,
which leads to failure
to hydroxylate
prolines and lysines
in collage
GLOBULAR PROTEINS: Tertiary
Structure and Functional
Diversity

Globular proteins not only possess

secondary structure but are also folded
into compact tertiary structures.
 The 3-dimensional arrangement of atoms
in the molecule.
In fibrous protein, backbone of protein
does not fall back on itself, it is important
aspect of 3 not specified by 2 structure.
In globular protein, more information
needed. 3k structure allows for the
determination of the way helical and
pleated-sheet sections fold back on each
other.
Interactions between side chains also
plays a role.
 Forces in 3 Structure
Noncovalent interactions, including
hydrogen bonding between polar side chains,
e.g., Ser and Thr
hydrophobic interaction between nonpolar side
chains, e.g., Val and Ile
electrostatic attraction between side chains of
opposite charge, e.g., Lys and Glu
electrostatic repulsion between side chains of
like charge, e.g., Lys and Arg, Glu and Asp
Covalent interactions: Disulfide (-S-S-) bonds
between side chains of cysteines
Forces That Stabilize Protein
Structure
 3and 4Structure

Tertiary (3) structure: the arrangement

in space of all atoms in a polypeptide
chain
it is not always possible to draw a clear
distinction between 2and 3structure
Quaternary (4) structure: the association
of polypeptide chains into aggregations
 Ubiquitin
has 76 amino acids
is synthesized in nearly all eukaryotic
cells
plays a critical role in targeting other
proteins for degradation
Varieties of Globular Protein Structure:
Patterns of Main-Chain Folding
Examples of diversity in protein tertiary structure
 Many proteins are made up of
more than one DOMAIN

A domain
is compact, locally folded region of
tertiary structure of roughly 150-250
amino acids
are interconnected by the polypeptide
strands that runs trough the whole
molecule
usually perform a function
 Domains may be composed of
repeating secondary structures, called
SUPERSECONDARY STRUCTURES
 Common Principles of Globular
Protein Structure
All globular proteins have a defined inside and
outside.
sheets are usually twisted, or wrapped into
barrel structures.
The polypeptide chain can turn corners in a
number of ways, to go from one segment or
helix to the next
Not all parts of globular proteins can be
conveniently classified as helix, sheet, or
turns.
Examples of beta turns
A gamma turn
 Myoglobin
A single polypeptide chain of 153 amino acids
A single heme group in a hydrophobic pocket
8 regions of -helix; no regions of -sheet
Most polar side chains are on the surface
Nonpolar side chains are folded to the interior
Two His side chains are in the interior, involved with
interaction with the heme group
Fe(II) of heme has 6 coordinates sites; 4 interact with
N atoms of heme, 1 with N of a His side chain, and 1
with either an O2 molecule or an N of the second His
side chain
The Structure of Myoglobin
 propionate

methyl methyl

vinyl
methyl

methyl vinyl

95
Oxygen Binding Site of Myoglobin
The geometry of iron coordination in
oxymyoglobin
 Oxygen-
binding
curve for
myoglobin
Quaternary Structure

Free template from www.brainybetty.com 99

 Quaternary (4) structure: the
association of polypepetide
monomers into multisubunit proteins
dimers
trimers
tetramers

Noncovalent interactions
electrostatics, hydrogen bonds,
hydrophobic
Oxygen Transport from
Lungs to Tissues:
Protein Conformational
Change Enhances
Function
Hemoglobins are tetramers (22) made up of
two kinds of myoglobin-like chains
Cooperative Binding and
Allostery
Changes in Hemoglobin
Structure Accompanying
Oxygen Binding
 Conformation Changes That Accompany Hb
Function
Structural changes occur during binding of
small molecules
Characteristic of allosteric behavior
Hb exhibits different 4 structure in the bound
and unbound oxygenated forms
Other ligands are involved in cooperative
effect of Hb can affect proteins affinity for
O2 by altering structure
Fig. 4-22, p. 102
108
 Oxygenation causes
hemoglobin quaternary
structure to change: One
dimer rotates and slide with
respect to the other
Iron lies 0.4Ao outside the protophorphyrin plane
in deoxyhemoglobin

110
The binding of oxygen allows iron to move into the plane
of the protoporphyrin ring.

111
On oxygenation, one pair of subunits shifts
with respect to the other by a rotation of 15o
Deoxyhemoglobin
(T sate)

Transition
 Allosteric Effectors of
Hemoglobin Promote Efficient
Oxygen Delivery to Tissue

H+, CO2, Cl- and 2,3-bisphosphoglycerate

are negative effectors of hemoglobin
 Response to pH Changes:
The Bohr Effect

Accumulation of CO2 lowers the pH in

erythrocytes through the bicarbonate
reaction:
CO2 + H2O HCO3-+ H+
A decrease in blood pH results
in stabilization of the deoxy
state and thereby favors
greater O2 released from
hemoglobin
This interaction locks the 1His146
to form salt bridge with 1Asp 94

119
 Carbon Dioxide Transport

A small portion of CO2 reacts directly

to hemoglobin to form carbamates:
122
124
Negative allosteric effectors
of hemoglobin act by
stabilizing the T-state
conformations
 Factors Determining
Secondary and Tertiary
Structure
Most of the information for
determining the three dimensional
structure of a protein is carried in
the amino acid sequence of that
proteins.
 Denaturation
Denaturation: the loss of the structural order (2, 3, 4, or a
combination of these) that gives a protein its biological
activity; that is, the loss of biological activity

Denaturation can be brought about by

heat
large changes in pH, which alter charges on side
chains, e.g., -COO- to -COOH or NH3+ to NH2
detergents such as sodium dodecyl sulfate (SDS) which
disrupt hydrophobic interactions
urea or guanidine, which disrupt hydrogen bonding
mercaptoethanol, which reduces disulfide bonds
Denaturation of a Protein
The Anfinsen
Experiment

Loss of Protein Structure: Because of small

differences between the free energy of folded and
unfolded proteins, they are susceptible to changes
in environmental factors
Denaturation and Refolding in Ribonuclease

Several ways to denature

proteins
Heat
pH
Detergents
Urea
Guanadine hydrochloride