The Three-Dimensional

structure of Proteins

Chapter 6
 The four levels of protein structure

 Primary (1˚) structure refers to the amino acid sequence that makes up
the protein
 Secondary (2˚) structure refers to local areas of repeating main chain
structure
 The four levels of protein structure

 Tertiary (3˚) structure refers to the spatial arrangement of the

secondary structural elements in the polypeptide chain
 Quaternary (4˚) structure refers to the spatial arrangement of multiple
polypeptide chains to form multisubunit complexes
Secondary Structure

• The secondary structure of a protein

describes repeating patterns of structure
within the three-dimensional structure of a
protein.

• The two most common secondary structures

are:
1. Alpha helix (helix)
2. Beta-pleated sheet (-pleated sheet)
• The  helix is a coiled structure, and much like the
coil of a telephone cord, it is a right-handed coil.

• This coil is stabilized by hydrogen bonds between

the carbonyl oxygen of one amino acid and the
N—H hydrogen atom of another amino acid located
four amino acids from it in the primary structure.

• The coil is able to stretch and recoil, and is a

strong structure. The side chains project outward
from the axis of the helix.
A secondary structure involves hydrogen bonding
along the backbone.
 The Alpha Helix
• Residues per turn: 3.6
• Rise per residue: 1.5 Angstroms
• Rise per turn (pitch): 3.6 x 1.5A = 5.4
Angstroms
• The backbone loop that is closed by any H-
bond in an alpha helix contains 13 atoms
• phi = -60 degrees, psi = -45 degrees
• The non-integral number of residues per turn
was a surprise to crystallographers
The -pleated sheet is an extended structure in
which segments of the protein chain align to form
a zigzag structure.
 The Beta-Pleated Sheet
Composed of beta strands
• Also first postulated by Pauling and
Corey, 1951
• Strands may be parallel or antiparallel
• Rise per residue:
•

– 3.47 Angstroms for antiparallel strands

– 3.25 Angstroms for parallel strands
– Each strand of a beta sheet may be
pictured as a helix with two residues per
turn
 The Beta Turn
(aka beta bend, tight turn)
• allows the peptide chain to reverse
direction
• carbonyl C of one residue is H-bonded
to the amide proton of a residue
three residues away
• proline and glycine are prevalent in
beta turns
• Strands called beta strands are held together
through hydrogen bonding interactions between
the backbone.

• The side chains of a -pleated sheet extend

above and below the sheet.

• The interactions of the side chains within the

secondary structure lead to the tertiary structure
of proteins.
 Side chain positions in an α helix

In an α helix, the side chains radiate away from the helical axis. The
center of the α helix consists backbone atoms, closely packed. The
hydrogen bonds that stabilize the helix are shown as yellow dashes.
 Side chain positions in a β sheet

In a β sheet, neighboring side chains are located on opposite faces of the

sheet, which is stabilized by main-chain hydrogen bonds between adjacent
β strands.
 Steric interactions determine peptide
conformation

Certain ϕ and ψ angles result in

steric clashes, where atoms are
closer than their van der Waals
radii. These conformations are not
allowed.
The backbone of an α helix
results in closely packed
atoms that do not
sterically clash.
 Ramachandran
plot

The Ramachandran plot

shows sterically allowed ϕ
and ψ angles. Shown here
are allowed angles for
poly-L-alanine in each of
various conformations.
White areas correspond
to sterically allowed
conformations based on
theoretical predictions.

The allowed regions would

be smaller
for amino acids with
larger R groups.
 Ramachandran plots for glycine and
proline
glycine proline

Proline (right) has a far more limited range of permissible angles than
glycine (left).
 Fibrous protein structure

• Fibrous proteins are elongated molecules with

well-defined secondary structures
• Examples include:
– keratin - hair, fingernails, feathers, scales, or
intermediate filaments (intracellular)
– fibroin – silk cocoons
– collagen - abundant connective tissue protein;
matrix material in bone on which mineral
components precipitate
 Alpha Keratin
• Found in hair, fingernails, claws, horns and
beaks
• Sequence consists of 311-314 residue alpha
helical rod segments capped with non-helical
N- and C-termini
• Primary structure of helical rods consists of
7-residue repeats: (a-b-c-d-e-f-g)n, where a
and d are nonpolar. Promotes association of
helices!
 α-keratin

In keratin, large hydrophobic residues repeat every

four positions; The α helix has 3.6 residues/turn,
giving each helix a hydrophobic side, which defines
the interface between two long helices in the
coiled-coil structure typical of keratin.
 Beta Keratin
Proteins that form extensive beta sheets
• Found in silk fibers
• Alternating sequence: Gly-
Ala/Ser-Gly-Ala/Ser....
• Since residues of a beta sheet extend
alternately above and below the plane of the
sheet, this places all glycines on one side and
all alanines and serines on other side!
• This allows Glys on one sheet to mesh with
Glys on an adjacent sheet (same for Ala/Sers)
 Collagen - A Triple Helix
Principal component of connective tissue
(tendons, cartilage, bones, teeth)
• basic unit is tropocollagen:
– three intertwined polypeptide chains (1000
residues each
– MW = 285,000
– 300 nm long, 1.4 nm diameter
– unique amino acid composition
 Collagen
The secrets of its a.a. composition...
• Nearly one residue out of three is Gly
• Proline content is unusually high
• Unusual amino acids found:
– 4-hydroxyproline
– 3-hydroxyproline
– 5-hydroxylysine
– Pro and HyPro together make 30% of res.
 Collagen
• Collagen, abundant connective tissue
protein; matrix material in bone, on which
mineral components precipitate; triple-
strand left-handed helix
• Contains hydroxyproline (Hyp) and
hydroxylysine
• G-X-Y tripeptide motif, where X is Pro
and Y is Pro or Hyp, lends itself to triple-
strand structure
• Polypeptide chains crosslinked and
glycosylated
• Vitamin C (ascorbic acid) is a cofactor
required for proline hydroxylation;
vitamin C deficiency (scurvy) leads to
collagen degeneration
 The Collagen Triple Helix
A case of structure following composition
• The unusual amino acid composition of
collagen is unsuited for alpha helices OR
beta sheets
• But it is ideally suited for the collagen triple
helix: three intertwined helical strands
• Much more extended than alpha helix, with a
rise per residue of 2.9 Angstroms
• 3.3 residues per turn
• Long stretches of Gly-Pro-Pro/HyP
Structural basis of the collagen triple helix

• Every third residue faces the crowded center

of the helix - only Gly fits here
• Pro and HyP suit the constraints of phi and psi
• Interchain H-bonds involving HyP stabilize helix
• Fibrils are further strengthened by intrachain
lysine-lysine and interchain hydroxypyridinium
crosslinks
Collagen
Three ways to represent
the 3D structure of the
small, single-chain protein
ubiquitin

Solvent-accessible surface model

Cartoon Stick model and close-up

Globular proteins fold into defined structures

 Proteins have diverse structures, with varying amounts of helix, sheet

and loop regions
 Larger proteins often contain two or more distinct “domains” of
compact folded structure
 A typical protein “domain” is ~200 amino acids and will fold
independently
 A domain frequently possesses some defined function (e.g., DNA
recognition, oligomerization, cofactor binding, etc.)
 Globular Proteins
Some design principles
• Most polar residues face the outside of the
protein and interact with solvent
• Most hydrophobic residues face the interior
of the protein and interact with each other
• Packing of residues is close
• However, ratio of vdw volume to total volume
is only 0.72 to 0.77, so empty space exists
• The empty space is in the form of small
cavities
 Common features of folded globular proteins

 Globular proteins have a nonpolar (hydrophobic) interior and a more

hydrophilic exterior
 β sheets are usually twisted or wrapped into barrel structures.
 The polypeptide chain can turn corners, e.g., β turns:
Distribution of hydrophobic and hydrophilic residues
in myoglobin
 Globular Proteins
The Forces That Drive Folding
• Peptide chain must satisfy the
constraints inherent in its own structure
• Peptide chain must fold so as to "bury"
the hydrophobic side chains, minimizing
their contact with water
• Peptide chains, composed of L-amino
acids, have a tendency to undergo a
"right-handed twist"
 Three thermodynamic factors influence folding
and stability of proteins
• Favorable intramolecular enthalpic interactions
– charge-charge interactions (ionic bonds; salt bridges)
– intermolecular hydrogen bonds
– van der Waals interactions; proteins are densely packed

• Unfavorable loss of conformational entropy

– Unfolded state has many conformations (high entropy)  Folded state
has only a few closely related conformations (low entropy)

• Favorable gain of solvent entropy from burying hydrophobic groups (the

“hydrophobic effect”)
– As hydrophobic side chains cluster in the interior they release ordered
solvent molecules from clathrate structures
Detail of H-bonding in a typical protein
Disulfide bonds  Bovine trypsin inhibitor: 3 –S-S– bonds

greatly increase  Effect is partly entropic; –S–S– reduce

stability the number of conformations possible in
the unfolded state

 Proteins with disulfide bonds primarily

exist and function extracellularly
 Intermediate and off-pathway states in protein folding

• Isomerization of prolyl bonds is catalyzed by peptide prolyl

isomerase; the cis isomer is disfavored by 1000-fold vs. the
trans isomer

• The formation of non-native disulfide bonds is corrected by

protein disulfide isomerase, which catalyzes the reduction and
reoxidation of disulfide bonds
Chaperones promote proper folding of proteins and thereby
prevent the formation of aggregated states associated with
disease
• https://www.youtube.com/watch?
v=d1QIEQEyYRo
Chaperonins: Protein complexes
that facilitate protein folding

The GroEL/ES chaperone of E. coli

Why needed?
1. Intracellular protein concentrations
are very high; GroEL/ES helps prevent
intermolecular interactions that lead
to protein aggregation
2. Protection needed when cell is heat-
stressed and proteins begin to unfold
 Schematic of chaperonin function

Interior is lined with After ATP and GroES bind, a

hydrophobic groups early conformational change occurs
in the process and the interior presents
hydrophilic groups – promoting
the proper folding of the
protein
 Several diseases involve misfolding of normal proteins
to give amyloid fibrils or amyloid plaques

Highly ordered amyloids form from non-native folding

intermediates or disordered aggregate states
Prions: Infectious agents that cause disease by inducing Amyloid fibrils of insulin
amyloid formation on contact
Prediction of secondary structure from amino acid
sequence

 Empirical methods are ~ 80% accurate

 Based on observed distributions of amino acids in helix vs. sheet

conformations (e.g., Ala prefers helix, Val prefers sheet, etc.)

 Amphiphilic α helix shows repeating patterns of side chain

polarity every 3-4 residues

 Amphiphilic β strand shows repeating patterns of side chain

polarity every other residue
Prediction of tertiary structure from amino acid
sequence

 Critical need for accurate

prediction from sequence,
since structures are known
for only ~ 1% of all known
sequences
 Prediction of tertiary
structure is difficult due to
the need to correctly predict
interactions between
residues that are far apart
in the primary structure
 Current computational
methods are ~ 60% accurate
SDS-PAGE (sodium dodecylsulfate
polyacrylamide gel electrophoresis)
 Chapter Summary
The Three-Dimensional Structure of
Proteins

The following are the four levels of protein

structure:
1. Primary (1o) refers to amino acid sequence.
2. Secondary (2o) consists of an alpha helix and a
beta-pleated sheet.
3. Tertiary (3o) refers to the folding of the 2o
structure.
4. Quaternary (4o) involves the interaction of
two or more polypeptides to form a biologically
active protein.
Denaturation of Proteins

• Denaturation of a protein disrupts the

stabilizing forces in the secondary, tertiary,
and quaternary structures.

• Denaturing agents consist of heat, pH, heavy

metal, organic chemicals, and mechanical
agitation.

• A denatured protein is not biologically active.

Examples of Biologically Important Proteins

• Collagen, hemoglobin, antibodies, and integral

membrane proteins are examples of
biologically important proteins.

• The biological function of proteins is dictated

by their structure.

• Changes in the structure of proteins affect

their biological function.