Chapter 4Protein Structure

Learning Outcomes:
1. Describe the structural levels of organization

in proteins and what types of bonds are

responsible for them.
2. Describe the basic features of alpha helices

and the two types of beta sheets.

3. Compare fibrous and globular proteins.
3. Describe and contrast the structure of

myoglobin and hemoglobin and the role of

conformational changes in the function of
hemoglobin.
4. Define native fold, denaturation and

refolding.
5. Summarize the effect of secondary forces on
conformations and protein folding.
 Levels of Protein Structure
1°structure: the sequence of amino acids in
a polypeptide chain, read from the N-terminal
end to the C-terminal end
2°structure: the ordered 3-dimensional
arrangements (conformations) in localized
regions of a polypeptide chain; refers only to
interactions of the peptide backbone

e. g.,α-helix and β-pleated sheet

3˚ structure: 3-D arrangement of all atoms
4˚ structure: arrangement of monomer
subunits with respect to each other
Protein with 585 residues
 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

Determination of 1˚ sequence is routine

biochemistry lab work. We will see this in
chapter 5.
 2˚ Structure-what
conformations are accessible

2˚ of proteins is hydrogen-bonded
arrangement of backbone of the
protein-this results in a regular repeat
unit when the protein folds
Two bonds have free rotation:
1)

1) Bond between α-carbon and amino

nitrogen in residue
2) Bond between the α-carbon and
 2˚ Structure-what
•
conformations are accessible

ω No rotation around omega

bond
 Structure of the Peptide
Bond
Structure of the protein is partially
dictated by the properties of the peptide
bond
The peptide bond is a resonance hybrid
of two canonical structures
The resonance causes the peptide bonds
be less reactive compared to e.g.
esters
be quite rigid and nearly planar
exhibit large dipole moment in the
favored trans configuration
The Rigid Peptide Plane and
the Partially Free Rotations
Rotation around the peptide bond, ω, is not
permitted
Rotation around bonds connected to the
alpha carbon is permitted
Φ (phi): angle around the α-carbon—amide
nitrogen bond
ψ (psi): angle around the α-carbon—
carbonyl carbon bond
In a fully extended polypeptide, both Φ and
ψ are 180°
Three bonds separate sequential a carbons in
a polypeptide chain. The N—Cα and Cα—C
bonds can rotate, described by dihedral
angles designated Φ and ψ , respectively.
The peptide C—N 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 groups.
A dihedral angle is the
angle at the intersection
of two planes defined by
bond vectors in the
peptide backbone

The atoms and planes

defining y .
By convention, Φ and
ψ are 180° (or –180°)
when the first and
fourth atoms are
farthest apart and the
peptide is fully
extended. As the
viewer looks out along
the bond undergoing
rotation (from either
direction) the Φ and ψ
angles increase as the
fourth atom rotates
clockwise relative to
the first
In a protein, some of
the conformations
shown here (e.g., 0°)
are prohibited by
steric overlap of
atoms. The balls
representing atoms
are smaller than the
van der Waal’s radii
for this scale.
ω ω Does not
rotate

ψ
Distribution of Φ and ψ
Dihedral Angles
Some Φ and ψ combinations are very unfavorable
because of steric crowding of backbone atoms with
other atoms in the backbone or side-chains
Some Φ and ψ combinations are more favorable
because of chance to form favorable H-bonding
interactions along the backbone
Ramachandran plot shows the distribution of
Φ and ψ dihedral angles that are found in a
protein
shows the common secondary structure
 Ramachandran Plot for L-Ala residues.
Peptide conformations are defined by the values of ϕ and
ψ.
Conformations deemed possible are those that involve
little or no steric interference, based on calculations using
known van der Waals radii and dihedral angles.
 Ramachandran Plot for L-Ala residues.
The areas shaded dark blue represent conformations that
involve no steric overlap and thus are fully allowed;
medium blue indicates conformations allowed at the
extreme limits for unfavorable atomic contacts;
the lightest blue indicates conformations that are
permissible if a little flexibility is allowed in the dihedral
 Ramachandran Plot for L-Ala residues.

The plots for other L residues for

unbranched chains are nearly
identical. Ranges for branched
residues are smaller,Val,
Ile,Thr.Plots for GLY are very broad.
Pro is very restricted.
Ramachandran Plot
Rabbit Pyruvate kinase
Most permissible
darkest blue-the
lighter the blue-
the less
permissible
(1963, 40 years
later Xray
structures
confirmed)
The values of ψ and Φ for all
the amino acid residues
except Gly in the enzyme
pyruvate kinase (isolated from
rabbit) are overlaid on the plot
of theoretically allowed
conformations. The small,
flexible Gly residues were
excluded because they
frequently fall outside the
expected (blue) ranges.
 α-Helix
Nature shows a profound right-
left asymmetry.

Right handed, alpha helices are

a consequence of using L amino
acids

D amino acids form left handed

helices
 α-Helix
Coil of the helix is clockwise or
right-handed

There are 3.6 amino acids per turn

Repeat distance is 5.4Å

Each peptide bond is trans and

planar
 α-Helix
C=O of each peptide bond is hydrogen
bonded to the N-H of the fourth amino
acid away

C=O----H-N hydrogen bonds are parallel

to helical axis

All R groups point outward from helix

α-Helix (Cont’d)
 α -Helix (Cont’d)
Several factors can disrupt an α -
helix

proline creates a bend because of (1)

the restricted rotation due to its cyclic
structure and (2) its α -amino group has
no N-H for hydrogen bonding
 α -Helix (Cont’d)
Several factors can disrupt an α -
helix
strong electrostatic repulsion caused by the
proximity of several side chains of like
charge, e.g., Lys and Arg or Glu and Asp
Several factors can disrupt an α -
helix
steric crowding caused by the proximity of
bulky side chains, e.g., Ser, Val, Leu, Ile,
Thr, Cys, Asn

Structures can also destabilize an alpha

helix if they are too small-glycine
 Beta -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 trans and

planar
 Beta -Pleated Sheet
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
 β -Pleated Sheet
Parallel
Antiparallel
Parallel beta sheets distribute nonpolar
residues on both sides of the beta sheet
Thus parallel beta sheets are core
structures
Antiparallel beta pleated sheets can
have all hydrophobic on one side and
hydrophilic on the other side.
Antiparallel beta sheets place
nonpolar residues on only one face of
the sheet
Only one face must be protected from
solvent
Thus antiparallel beta sheet proteins
may contain as few as two layers
 β -Pleated Sheet
Parallel
Antiparallel
β -turn in antiparallel
pleated sheet

In globular proteins with compact strudtures

about 1/3 of the residues are in turns where
the polypeptides reverse directions.
Beta -Pleated Sheet
Type I and type II β turns are most common;
type I (with proline) turns occur more than
twice as frequently as type II.
Type II b turns usually have Gly as the third
residue in reverse turn.
Note the hydrogen bond between the peptide
groups of the first and fourth residues of the
bends. (Individual amino acid residues are
framed by large blue circles.)
 β -Pleated Sheet

β-bulge- a common non-repetitive

irregular 2˚ structure in anti-parallel
structure
Schematic Diagrams of
Supersecondary Structures also Called
Motifs or Domains
 α-Helices and β-Sheets
Supersecondary
structures:
the combination of α- and –
β sections.
Schematic Diagrams of
Supersecondary Structures βαβ
unit: two parallel strands of b-sheet connected
by a stretch of α-helix
Schematic Diagrams of
Supersecondary Structures αα
unit: two antiparallel α-helices
Schematic Diagrams of
Supersecondary Structures β-
meander: an antiparallel sheet formed by a series
of tight reverse turns connecting stretches of a
polypeptide chain
Schematic Diagrams of
Supersecondary Structures Greek
key: a repetitive supersecondary structure
formed when an antiparallel sheet doubles back
on itself
Schematic Diagrams of
Supersecondary Structures β-
barrel: created when β-sheets are extensive
enough to fold back on themselves
Supersecondary structure, domain
and motif are used interchangeably.
Often correspond to a specific
function
For example, the β-barrel,
surrounded by α-helices is known
as the TIM domain named for
triosephosphate isomerase and is
found in the active site of the
enzyme.
 Tertiary Structure 1.Fibrous
Proteins
Fibrous proteins: contain polypeptide chains
organized approximately parallel along a single
axis. They
consist of long fibers or large sheets
tend to be mechanically strong
are insoluble in water and dilute salt solutions
play important structural roles in nature
Examples are
keratin of hair and wool
collagen of connective tissue of animals
including cartilage, bones, teeth, skin, and
blood vessels
 Structure of Collagen

Collagen is an important constituent

of connective tissue: tendons, cartilage,
bones, cornea of the eye
Each collagen chain is a long Gly- and
Pro-rich
left-handed helix- Distinct from alpha helix
Three collagen chains intertwine into a
right-handed superhelical triple helix
Structure of Collagen

Why left handed helices?

Insoluble in water, lots of hydrophobic
glycine and proline
Glycine is achiral
Many peptide bonds in pro are cis
No hydrogen donor in proline peptide bond
-X-Pro-Gly and -X-Hyp-Gly (hydroxyproline)
sequences
J. Mol. Biol. (2005) 347, 231–241
Structure of Collagen- Proline
 Tertiary Structure 2.Globular
Proteins
Globular proteins: proteins which are folded to
a more or less spherical shape
they tend to be soluble in water and salt
solutions
most of their polar side chains are on the
outside and interact with the aqueous
environment by hydrogen bonding and ion-
dipole interactions
most of their nonpolar side chains are buried
inside
nearly all have substantial sections of α-helix
and β-sheet
Comparison of Shapes of Fibrous and
Globular Proteins
 3˚ Structure
The 3-dimensional arrangement of atoms in the
molecule.

In fibrous protein, backbone of protein does not

fall back on itself, and the only aspect of 3˚ not
specified by 2˚ structure is the arrangement of
side chain atoms.

In globular protein, more information needed. 3-D

structure specifies 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
Metal ion coordination
Forces in 3˚
Structure
 3°and 4°Structure
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 2°and 3°structure

Quaternary (4°) structure: the association of

polypeptide chains into aggregations

Proteins are divided into two large classes based

on their three-dimensional structure
fibrous proteins
globular proteins
Globular Protein :Myoglobin
found in muscle tissue in vertebrates

A single polypeptide chain of 153 amino acids

A single heme group in a hydrophobic pocket
8 regions of α-helix; no regions of β-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
Oxygen Binding Site of Myoglobin
 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 and Refolding in
Ribonuclease

Several ways to denature

proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride
Mercaptoethanol
Denaturation and Refolding in
Ribonuclease
Denaturation and Refolding in
Ribonuclease
Chapter 3 Amino Acids and
Peptides

Cysteine is oxidized and forms

hydrophobic S-S bonds
Denaturation and Refolding in
Ribonuclease
 Quaternary Structure
Quaternary (4°) structure: the
association of polypepetide
monomers into multisubunit proteins
dimers
trimers
tetramers

Noncovalent interactions
electrostatics, hydrogen bonds,
hydrophobic
Oxy- and Deoxyhemoglobin

Deoxyhemoglob
in
Oxyhemoglobin
:
Less room
Oxygen Binding of Hemoglobin
(Hb)found in red blood cells
A tetramer of two α-chains (141 amino acids each) and
two β-chains (153 amino acids each); overall, α2 β2
Each chain has 1 heme group; hemoglobin can bind up
to 4 molecules of O2
Binding of O2 exhibited by positive cooperativity;
when one O2 is bound, it becomes easier for the next
O2 to bind
The function of hemoglobin is to transport oxygen
The structure of oxygenated Hb is different from that
of unoxygenated Hb
H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect
the ability of Hb to bind and transport oxygen
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
protein’s affinity for O2 by altering
structure
Myoglobin plots of
% saturation
versus partial
pressure of oxygen
are hyberbolic. The
bonding curve for
hemoglobin is
sigmoidal. The
sigmoidal curve
indicates
cooperativity
The effect of secondary forces
on conformations and protein
folding.
Conformations change by rotating bonds.
Secondary forces restrict the conformations that
are accessible to proteins. Examples are seen in
beta pleated sheets and alpha helices.
The driving force for protein folding is the
hydrophobic effect. Entropy increases as the
surface areas of hydrophobic structures are
minimized. This increases the entropy of the
surrounding water molecules.