Charter 2: Protein Composition and Structure

X-ray crystallography
Nuclear Magnetic Resonance, NMR
Cryo-Electron Microscopy

Protein Structure Prediction, AlphaFold

 Learning Goals
By the end of this chapter, you should be able to:

• Identify the 20 amino acids and their corresponding three-letter and one-letter abbreviations, and
group them according to the chemical properties of their side chains.

• Distinguish between primary, secondary, tertiary, and quaternary structures.

• Describe the properties of the principal types of secondary structure, including the α helix, the β sheet,
and the reverse turn.

• Explain how the hydrophobic effect serves as the primary driving force for folding of polypeptide
chains into globular proteins.

• Describe the nucleation-condensation model of protein folding, and explain why it is preferable to a
random sampling model.
 Outline

2.1 Several Properties of Protein Structure Are Key to Their Functional Versatility

2.2 Proteins Are Built from a Repertoire of 20 Amino Acids

2.3 Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains

2.4 Secondary Structure: Polypeptide Chains Can Fold into Regular Structures

2.5 Tertiary Structure: Proteins Can Fold into Globular or Fibrous Structures

2.6 Quaternary Structure: Polypeptide Chains Can Assemble into Multisubunit Structures

2.7 The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure
2.1 Several Properties of Protein Structure Are Key to
Their Functional Versatility: 20x

Space-filling model Ribbon model

DNA replication clamp

Bacterial beta clamp

Proteins are linear polymers built of monomer units called amino acids.

•The amino acid sequence of a protein dictates its folding process.

•The 3D structure of a protein determines its biological function.

 Rules for 3D Structural Foldability.

1. Proteins contain a wide range of functional groups.

2. Proteins can interact with one another and with other biological
macromolecules to form complex assemblies.

3. Some proteins are quite rigid, whereas others display considerable

flexibility.
•Structural elements are rigid. Why?
(cytoskeletons, connective tissues, etc.)
•Regulatory elements are flexible. Why?
(protein-protein interaction, signal transduction, etc.)

Conformational Changes of

Lactoferrin upon iron binding

2.2 Proteins Are Built from a Repertoire of 20 Amino Acids

Central

Only L-amino acids are

constituents of proteins.
Zwitterionic Character of Amino Acids (Dipolarity)
 Amino Acid Nomenclature

Proteins are built from a repertoire of 20 amino acids in all species.

Twenty kinds of side chains vary in size, shape, charge, hydrogen bonding
capacity, hydrophobic character, and chemical reactivity.
 Classification of Amino Acids
Based on the Characteristics of Functional Groups

Polar (Hydrophilic) Non Polar (Hydrophobic)

Arg, Asn, Asp, Cys, Ala, Gly, Ile,
Gln, Glu, His, Lys, Leu, Met, Phe,
Ser, Thr, Trp, Tyr Pro, Val

Acidic (negative charge) Basic (positive charge)

Asp, Glu Arg, Lys, His

Aromatic (ring) Aliphatic (linear chain)

Phe, Tyr, Trp Most of the rest
Building blocks of proteins
 achiral
(non-chiral)

Ball-and-stick
model
Most
Simple
Perspective Amino
drawing
Acids

Structural
formula
 Most
Typical
Aliphatic
Amino
Acids

Most
Typical
Non-Polar
Hydrophobic
Amino
Acids
 The side chain of proline is bonded to
both the nitrogen and a-carbon atom.

Proline is an imino acid.

Structural flexibility is much more restricted than other amino acids.
Proline markedly influences protein architecture.
 Most
Typical
Aromatic
Amino
Acids

The hydroxyl group

in tyrosine is
chemically reactive.

Tryptophan contains
an indole ring.

The aromatic rings of

Tyr and Trp contain
delocalized p electrons
absorbing UV light.
 Tryptophan and Tyrosine Can Be Useful
for the Determination of Protein Concentration

Beer’s Law : A = ecl

A : Absorbance,
e : extinction coefficient (M-1cm-1),
c : concentration (M),
l : length of light pass (cm)

Maximum absorbance
at 276 nm for Tyrosine
at 280 nm for Tryptophan
 Ser and Thr
Contain
Aliphatic
Hydroxyl Group.

Ser is more
hydrophilic than Ala.

Thr is more
hydrophilic than Val.

Threonine
Contains
an Additional
Chiral
Carbon
 Cysteine is structurally similar to serine but contains
a sulfhydryl, thiol (-SH), group in place of the hydroxyl (-OH) group.

Pairs of sulfhydryl groups can form a disulfide bonds

which can be critical in stabilizing three dimensional structure in some proteins.
Very Polar, Highly Hydrophilic, and
Positively Charged Amino Acids

Lys : e-amino group

Arg : guanidium group
His : imidazole group

Histidine Ionization
 Very Polar,
Highly Hydrophilic,
and
Negatively Charged
Amino Acids
 Asparagine (Asn)
Uncharged Derivatives of Aspartate
b-carboxamide group

Glutamine (Gln)
Uncharged Derivatives of Glutamate
g-carboxamide group
Seven Amino Acids
Containing
Readily Ionizable
Side Chains

Aspartate
Glutamate
Histidine
Cysteine
Tyrosine
Lysine
Arginine
 pKa values of
functional groups
in actual proteins
can be dramatically
changed by the
microenvironment
where
the given side chains
are located !!!
2.3 Primary Structure:
Amino Acids Are Linked by Peptide (Amide) Bonds
to Form Polypeptide Chains (Proteins)

First Second
Amino Acid Amino Acid
water loss

•The formation of a peptide bond requires an input of free energy

•But, the peptide bond is very stable once it is formed.
(T1/2 in aqueous solution : 1000 years, hydrolysis rate is so slow)
•The order (sequence) of amino acids in a polypeptide chain is called
the primary structure of a protein.
 Backbone or Main Chain
Amino acid sequence have direction (Regularly Repeating Part)
vs.
Functional Group or Side Chain
(Variable Part)

One Amino Acid in a Protein

Is Called as a Residue.

Directionality of Polypeptide Chain

N-terminus  C-terminus
(YGGFL ≠ LFGGY)

Alternative Positioning of
the Oxygen and the Hydrogen
in One Peptide Bond

Alternative Positioning of
the Oxygen and the Hydrogen
between Neighboring Peptide Bonds

Alternative Positioning of
the Functional Groups
Between Neighboring Residues
 Disulfide Bonding

• In some proteins, the linear polypeptide chain can be cross-linked and the most
common cross-links are disulfide bonds between cysteine residues.
• Extracellular proteins form disulfide bonds more often than intracellular proteins.
FIGURE 2.16
The primary structure of bovine insulin is its amino acid sequence.
 Size of Polypeptide Chain
• Most natural polypeptide chains contain between 50 and 2000
amino acid residues and are commonly referred to as proteins.
• Less than 50 amino acids  oligopeptides or peptides
• The average molecular weight of an amino acid is about 110 Dalton.
Thus, the molecular weights of most proteins range between 5500
and 220000 dalton (i.e. 5.5 kd to 220 kd).

Amino Acid Sequences of Proteins

• Each protein has a unique and precisely defined amino acid sequence.
• Central Dogma : DNA  RNA  Protein
• Amino acid sequence of a protein determines its structure, function,
and the mechanism of biological action.
• Changes in amino acid sequences  Disease, Genetic Engineering
Properties: Polypeptide chains are flexible yet conformationally restricted

Typical Bond Lengths

within a Peptide Bond

Peptide bonds are planar

The peptide bonds contain

• Considerable double bond character
• High H-bond forming capacity to proteins.
(Peptide Bond  Peptide Bond, or
Peptide Bonds  Functional Groups)
The peptide bond resonates between a single BUT,
bond (1.49Å) and a double bond (1.27Å).
still uncharged  tightly packed structure
 Most peptide bonds have a trans configuration.

a-Carbons

>

Trans-Configuration of a-Carbons around a Normal Peptide Bond

cis bonds are as likely as trans bonds for proline and its preceding residues.
 Rotational Properties of Peptide Bonds

Peptide bonds are rigid…

But,
the bonds containing the a-carbon between two peptide bonds
can be rotated from -180o to +180o.

, phi : the angle of rotation about the bond between the nitrogen and the a-carbon
y, psi : the angle of rotation about the a-carbon and the carbonyl carbon
Ramachandran Diagram: Rotational Properties of Peptide Bonds

• Ramachandran Diagram Shows the Allowed Ranges of  and y Rotations due to collisions.
• For Some Combinations of  and y Rotations Are Physically Impossible due to Steric Clashes.
• Protein folding is possible by rigidity of peptide unit and restriction of  and y Rotations
2.4 Secondary Structures: Polypeptides Chains Can Fold
Into Regular Structures

Alpha Helix, Beta Pleated Sheet, Turns, Loops

Linus Pauling and Robert Corey’s proposal – 1951

The Double helix – 1953 James D. Watson
 The a-Helix is a Coiled Structure
Stabilized by Intra-Chain Hydrogen Bonds
• Rise of 1.5 Å per Residue
along the Helix Axis
• Rotation of 100 degree per
Residue around the Helical
Turning
• 3.6 Amino Acids (aa) per a
Single Turn of a-Helix
• Thus, amino acids spaced
three to four residues apart
are spatially quite close to
one another in an a-helix.
4 residues
H-bonds forms
between the CO group residue i
and NH group of residue i+4
in peptide bonds of a-helix
 Coiling and Entwining of a-Helix

• Essentially all a-helices in proteins are right handed.

(Ramachandran diagram explains it why.)
• The a-helical content of proteins range from none
to almost 100%.
• Single a-helices are usually less than 45 Å long.
• Two or more helices can be entwined and form a
very stable and long coiled coil structure with a Probability of
length of 1000 Å long a-Helix Coiling Direction

a-helical coiled coil; superhelix;

tropomyosin, keratin, fibrin;
ribbon and rod Ferritin contains 75% of a-helices. bundles of fibers; filamentous structures
(cylindrical) depiction
 Beta Pleated Sheet

• Almost Fully Extended Structure

• Distance between adjacent amino acids is 3.5 Å .

• The side chains of adjacent amino acids point in

opposite directions.

• H-bonding between different b-strands Combinations of  and y rotations

allowing the formation of b-sheet
• Why beta?
Two Simplest b-Sheet Structures

Anti-Parallel b-Sheet

H-Bondings
between
Single Amino Acids

Parallel b-Sheet

Overlapped H-Bondings
between
Two Amino Acids
More b-Sheet Structures

Twisted b-Sheet Structure

with Multiple b-Strands

An Example of
a Protein
Rich in b-Sheet

Fatty Acid
Mixed b-Sheet Structure Binding Protein
with Multiple b-Strands
 Turns and Loops

• Omega Loop ( loop) also

enables reversals in the direction of
polypeptide chains.
• Reverse Turn (b-turn, hairpin bend) • No regular and periodic structures
enables reversals in the direction of • But, loop structures could also be
polypeptide chains. rigid and well defined.
• These reversals allow proteins to form • Invariably located on the surface
compact and globular structures. • Protein-protein interactions
2.5 Tertiary Structures: Proteins Can Fold
Into Globular or Fibrous Structures
 What is Van der Waals force?

 Weak electric forces between neutral molecules by fluctuating polarization of

nearby particles

 3 source; permanent dipole-permanent dipole forces, permanent dipole-

induced dipole force, Instantaneous induced dipole-induced dipole (London
dispersion forces)

 Named after Dutch physicist, Johannes Diderik van der Waals

Nobel prize winner in physics in 1910
 van der waals interaction
It is caused by transient dipoles, the momentary random fluctuation in
the distribution of the electrons of any atoms
- 1/r6 dependence
Hydrophobic Effects?

Stabilized by van der

waals interaction
1-4. Bonds that Stabilize Folded Proteins
Folded proteins are stabilized mainly by weak noncovalent interactions

1 kcal = 4.2 kJ
Figure 1-10 Table of the typical chemical interactions that stabilize polypeptides
 General Rules of Protein Folding

• In an aqueous environment, protein folding is driven by the strong tendency of

hydrophobic residues to be excluded from water.- called hydrophobic effect.
• Contrasting distribution of polar and non-polar residues: the hydrophobic side
chains are buried inside, whereas the hydrophilic and charged functional groups
are headed to the outer surface.
• All the NH and CO groups from the interiorly located peptide bonds holding non-
polar side chains (i.e. peptide bonds around hydrophobic environment) are forced
to form hydrogen bonds.
• Therefore, these multiple hydrogen bonding enhance the interior structural integrity
by efficiently establishing a-helix and b-sheet structures.
• Van der Waals interactions between hydrophobic side chains also contributes to
the structural stability of a protein.
 Tertiary (3rd) Structure
3D Structure of Myoglobin

• Water-soluble proteins fold into Ribbon model

compact structure with nonpolar

cores
• Three dimensional structure of a
polypeptide chain – grouping of
amino acids
Space-filling model
• Generally, protein folding yields very
compact tertiary structures (10 fold). 1) 7 a-helices (70% of main chain) are linked by turns
and loops.

2) Heme = Protoporphyrin + Iron; Prosthetic Group;

Oxygen Binding
 Key Aspects of Myoglobin 3D Structure

Hydrophobic aa

Charged aa

Surface cross section

• The interior space consists almost entirely of non-polar residues.

(e.g. Val, Leu, Met, Phe, etc.)
• The charged residues are absent from the inside of a protein.
(e.g. Asp, Glu, Lys, Arg, etc.)
• The only polar residues inside are two His; iron and oxygen binding
• The surface outside consists of both polar and non-polar residues.
• There is very little free empty space inside.
Inside-Out Folding: Exception of Protein Folding

Membrane protein, Porin

• Proteins found in the outer membranes of

many bacteria
• The outside is covered with hydrophobic
residues interacting with neighboring alkane
chains. (cf. permeability barriers of the
biological membranes)
• The center of the protein contain a water-
filled channel lined with charged and polar
amino acids.
• Hydrophobic vs. Aqueous Environment
“Inside out”
• Membrane vs. Cytosolic Proteins
 Motif
•Certain combinations of secondary structures
with similar functions

• Functional supersecondary structure

•DNA binding proteins

Four level of structural organization

•Primary structure: the amino acid sequence

•Secondary structure: spatial arrangement of a.a nearby in sequence, a helix and b strand

•Tertiary structure: spatial arrangement of a.a far apart in sequence.

 Domain
• A compact and globular structural unit of a protein is often called as a domain
(i.e. pearls on a string)
• The size of a domain ranges from 30 to 400 amino acid residues.
• Different proteins can have a similar or the same domain.
• Domain is a structural working unit of a protein for the common function.

4 Domains in CD4 ; Each domain with approximately 100 Amino Acids

 Coiled-coil protein
• Structural support for Cells and Tissues
 a-keratin: left-handed superhelix of two right-handed a helices.
from wool & hair, intermediate filaments in cytoskeleton, muscle protein (myosin & tropomyosin)
 Heptad repeats; Every seventh residue in each helix, Leu holds two helix by van der Waals
interactions that stabilize close contacts between hydrophobic nonpolar surfaces.
 disulfide bond crosslinks: fewer – flexible, more – harder (horns, claws etc)
• Collagen: the most abundant protein of mammals, main fibrous component
of skin, bone, tendon, cartilage, and teeth. (Skin Care)

at every 3rd residues

d=15 Å

L=3000 Å = 1000 aa

Superhelical cable by triple-strands

2.6 Quaternary Structure: Polypeptide Chains Can
Assemble into Multisubunit Structures
 Subunit
each polypeptide chain in a protein containing more than one polypeptide chain

Quaternary Structure
The spatial arrangement of subunits and the nature of their interaction

human rhinovirus
(common cold)

Cro
(Bacteriophage )
a dimer of Hemoglobin Viral coat protein
identical subunits Oxygen-carrying protein 60 copies of each
(homodimer) Hetero – Tetramer (a2b2) of 4 subunits
with 4 Heme groups (240 polypeptide chains)
2.7 The Amino Acid Sequence of a protein Determines Its
Three-Dimensional Structure

Denaturant

Ribonuclease (124 AA; 4 Disulfide Bonds)

Reductant

Christian Anfinsen in the 1950s

 A Lesson from Ribonuclease Observed by Anfinsen

8M urea
b-mercaptoethanol

slow dialysis &

oxidation  remove b-mercaptoethanol
slow refolding & first and then remove urea
regaining
activity

trace
amounts of
b-mercapto
ethanol

Random coiled ribonuclease

scrambled

The information needed to specify the catalytically active structure of ribonuclease

is contained in its amino acid sequence : Sequence specifies conformation !!!
Each amino acid has its own preference
a-Helix could be default.
to form a-helix, b-sheet, or turns.
Val and Ile prefer
b-sheet due to
their branching at b-carbon.

Pro breaks
both a-helices and b-sheets
due to its ring structure.

Ser, Asp, Asn often disturb

the formation of
a-helix due to their
capability
to easily form
extra hydrogen bonds
with various side chains.
Many sequences can adopt alternative conformations
How a.a. sequence specify protein structure?
How an unfolded polypeptide chain acquire the native tertiary structure?
How about secondary structure?

VDLLKN in a-helix VDLLKN in b-strand

In many cases,
the context is very crucial in determining the conformational outcome.
(cf. the accuracy of predicting secondary structures using oligopeptides < 60 to 70%)
Cooperativeness in Protein Folding : ALL or NONE Process

Cooperative
and Sharp
Transition

1:1 Mixture (Folded &

Unfolded Proteins),
no half-folded protein

heating, urea or guanidinium chloride

The proposed folding pathway of chymotrypsin inhibitor-2
demonstrates the nucleation-condensation model
 Protein folding funnel
Thermodynamics of protein folding
 Computational prediction of folding is now reliable

• Ab initio method
- Equilibrium conformation is the global free-energy minimum
- potential energy parameter is accurate (H-bond, van der Waals etc)
- oligomerization can not be addressed although very many globular proteins are oligomeric.
- The methods is limited by the vast number of possible conformations, the marginal stability of
proteins, and the subtle energetics of weak interactions in aqueous solution

• AlphaFold and RoseTTaFold Critical Assessment of Structure Prediction (CASP)

- Deep Learning-Based: Both use advanced neural networks to predict protein structures.
- Integration of Multiple Data Sources: They combine evolutionary, structural, and sequence data to
improve prediction accuracy.
- End-to-End Model: AlphaFold provides an end-to-end approach, directly predicting 3D structures
from amino acid sequences.
- Flexibility and Efficiency: RoseTTaFold is designed to handle multiple sequence alignments and
can predict protein-protein interactions, making it versatile for various structural biology tasks.
 CASP competitions in 2020.

The AlphaFold algorithm (red bar) predicted the structure with nearly 90% accuracy
 Protein misfolding and aggregation are associated with
some neurological diseases

The structure of the Aβ amyloid fibril reveals The extent of amyloid fibril aggregation in the brain
its extensive β sheet network can predict the progression of Alzheimer disease.
Posttranslational modifications (PTM) confer new capabilities to proteins
• Progressive Stabilization of Intermediates during Folding rather than random search
• Prediction of Three Dimensional Structure from Amino Acid Sequence?
- ab initio prediction
- knowledge-based methods
• Post-Translational Modification
- Phosphorylation: serine, threonine, and tyrosine, signaling switch
- Glycosylation: Asn (N) and Ser and Thr (O-GlcNAc), solubility increase and protein-protein interaction
- Acetylation: N terminal of proteins, resistant to degradation.
- Hydroxylation: hydroxylation of proline in collagen stabilization, Vitamin C deficency
- Carboxylation: glutamate in prothrombin, Vitamin K deficency - hemorrhage
- Acylation: additon of a fatty acid to a-amino group or cysteine sulfhydryl group
-Carbamylation
 Green fluorescent protein (GFP)
•composed of 238 amino acids (26.9 kDa),
originally isolated from the jellyfish Aequorea
victoria

•fluorescens green when exposed to blue light

•Used as a reporter of expression & biosensor

•The GFP gene can be introduced into

organisms (bacteria, yeast and other fungal
cells, plant, fly, and mammalian cells)

•2008 Nobel Prize in Chemistry : Martin

Chalfie, Osamu Shimomura and Roger Y.
Tsien Fluorescence of GFP chromophore by
cyclization reaction including rearrangement
•A typical beta barrel structure and oxidation on Ser-Tyr-Gly.

 Cleavage after protein synthesis

- digestive enzymes (pancreas, intestine)
- blood clotting factor (fibrinogen  firbrin)
- hormone, viral proteins