Section 5-5 National Tsing Hua University

CHEM5980

Protein hierarchical structures

• Primary structures are sequence of

protein
• Secondary structures are α helix, β
sheet and turns.
• Domains are small regions of protein
capable of folding independently that
formed by interaction between α helix
and/or β sheet. Typically less than 100
amino acids.
• Tertiary structure is consist of one or
several domains.
• Quaternary structure is assembled from
multiple independent protein chains to
form complex structures. They are
usually nanometer sized machines.
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CHEM5980

Alpha helix
• Hydrogen bonds can form within a peptide chain between the carbonyl oxygen and
the amide hydrogen. As the such groups on peptide backbone are regularly
positioned. It is possible for them to form an arranged structure with repetition.
• To investigate such intramolecular hydrogen bond systematically, interactions
between carbonyl oxygen and amide hydrogen are evaluated.
• Hydrogen bonding between the same residue (both i), neighboring
residues (i and i+1) and skipping residues (i and i+2) are not energetically
favorable.
• Hydrogen bonding between i
and i+3 residue result in 310
helix, however the strain
prevent it longer than few
residues.
• In contrast i and i+4 residue
form stable α helix, which is
highly polarized due the the
alignment of the hydrogen
bonds toward the helix axis.
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Alpha helix wheel

• α-helix has 3.6 amino acid residues per turn and 1.5 Å rise along axis per residue,
such uniform structure allows a more concise way to represent its structure.
• If viewing along the helical axis, the end closer to observer has larger diameter and
can be represented in the larger diameter circle. As the sequence goes further,
residues away from observer becomes smaller and drawn on a smaller circle.
• The plot reveals the location of amino acid residue on which side of the helix, to
judge the interaction with other secondary structures. Normally the hydrophobic
amino acids are concentrated on one side of the helix while polar or hydrophilic
amino acids on the other.

Met

Met

α-helix viewing
from top
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CHEM5980

Beta sheets
• β sheet is the another common form of regular secondary structure in proteins.
• Hydrogen bonds between peptide backbones stabilizes the structure. There are
two possibilities for the direction of the peptides chains that involved. Antiparallel
β sheet has the neighboring interacting peptides in different directions whereas
parallel β sheet has peptides in the same direction.
• β sheet is not flat and forms repeatedly pleated plates. It is also common for the
formation of β barrel.

β-barrel that
allows sucrose to
pass through
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CHEM5980

Turn structure
• α helix and β sheet both are directional. For a protein to
form globular structure, it is necessary for the peptide chain
to change direction.
• The point on the peptide chain to make direction change is
called turn structure, which has minimal backbone hydrogen
bonds and is always found at the exterior of protein to
contribute to protein binding interactions.
• β turn involves hydrogen bonding between ith and i+3th
residues and forms hairpin. In contrast, γ turn has hydrogen
bond between ith and i+1th residues and turns right angle.
• The likelihood for amino acid residue to involve in different
secondary structures are listed in the table.
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CHEM5980

Disulfide bonds in protein

• The fastest disulfide bonds formed after
translation are not necessary the native
one. Inclusion body is the aggregation of
misfolded protein that requires refold.
• If the glutathione concentration is high
enough, the disulfide bonds are allow to
quick exchange to refold. But if the
concentration is too high, the disulfide
bonds are stable.
• Heterologous expression
(human protein in bacteria)
in non-native organism
usually lead to misfolding. In
vitro refolding requires thiol
concentration to be tested
for best result.
• The total concentration of
glutathione is 5mM in most
cells, but the GSH and GSSG
ratio differs depend on the
location.
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CHEM5980

Protein domains
• Protein domains are modular units with specific arrangements of secondary
structures. Sometimes they are stable without the rest part of protein and with
original functions.
• Protein domains can be reused for different functions, this protein evolution can be
done by homologous recombination.
• Some domains are
popular in proteome,
but the extent may
be different from
organism to organism.
Common protein
domains in human
proteome are listed.
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CHEM5980

Zinc finger domain

• Classical zinc finger C2H2 (2 Cys and 2 His) is the most common domain in human
proteome. It stabilize the folding by the coordination of a zinc ion.
• Classical zinc finger is about 25 amino acid residues with an α helix and a peptide loop.
One C2H2 zinc finger variant has 2 cysteine residues on the loop and 2 histidine
residues on helix (X2-Cys-X2,4-Cys-X12-His-X3,4,5-His) to coordinate to zinc ion.
• Classical zinc finger proteins bind DNA in major groove with specificity as nucleases
and transcription factors. Zinc finger is important in DNA sequence recognition. The
occurrence of zinc finger in human proteome indicates the importance of transcription
factor in human cells.
• A broader definition of zinc finger is
structural motif the bind zinc to stabilize the
structure. there are a number of unique
types of zinc fingers, each with a unique
architecture.
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CHEM5980

β-sandwich architectures
• Immunoglobulin domain composed of two face-to-face anti-parallel β sheets, which
consist of 3~4 β strands.
• The individual β strands connect back and forth in both sheets; as an example, one
sheet may contain β strand in order of strand 1, 2, 5 and 4 strand and the other
sheet has strands 3, 6, and 7.
• In immunoglobulins, the immunoglobulin domains are connected by flexible hinge
regions, which functions as joints for the arm. This morphology allows adjustable
bidentate adjustment to bind to antigen.

Immunoglobulin type G (IgG)

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β-sandwich architectures
• Immunoglobulin domains also present in proteins other
than immunoglobulin, such as muscle protein, in which
the immunoglobulin domain contribute to the its
resiliency property.
• Titin is the third abundant protein in muscle that limits
the range of motion of the sarcomere in tension. The
immunoglobulin domains unfold when the protein is
stretched and refold when the tension is removed

Circulation 2006, 113, 1922

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CHEM5980

β-sandwich architectures
• Fibronectin is a glycoprotein of the extracellular matrix that binds components such
as collagen, fibrin, and heparan sulfate proteoglycans.
• Fibronectin type III domain is another popular in human proteome with anti-
parallel β sandwich structure.
• Olfactory cells adhesion molecule (OCAM) plays a role for compartmentalization of
synapses within the glomerular layer. OCAM binds to each other through a
immunoglobulin domain and provides cell adhesion. Both immunoglobulin domains
and Fibronectin type III domains (Fn3I and Fn3II) are available in OCAM.

OCAM

OCAM
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β-sandwich architectures
• Cadherin domain coordinate adhesion between cells through protein complex
anchored to internal cytoskeleton actin.
• The extracellular part of cadherin contains 5 cadherin folding domains. The terminal
domain can dimerize with another similar domain from another cell.
• During dimerization, tryptophan residue on one cadherin domain binds to
hydrophobic pocket of the complementary domain and results in β strand exchange.
• Cadherin requires calcium ion to support its structure although it only weakly binds
to cadherin.
• Losing cadherin on cancer cells are related to invasive tumor.
Ca2+
Trp
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CHEM5980

WD domains
• WD domain is formed with 4-strand anti-parallel β sheet that with a orthogonal
twist over 40 amino acids and therefore called WD40 domain.
• The WD40 domain is composed of several repeats, a ~20-residue variable region of
is followed by a more common repeated set of residues.
• Blade-like WD40 domain is formed and fit together with other WD40 to form a
propeller-like structure with the 7 bladed beta propeller being most common.
• WD domain serve as diverse roles in cell signaling, including regulation of
transcription, cell-fate determination, trans-membrane signal transduction, vesicle
trafficking and others.

Trend Biochem Sci 2007, 32, 547

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CHEM5980

Three-strand helix of collagen

• Collagen is the most abundant protein (25-35%) in human and can be found in
extracellular matrix.
• Collagen is exploited as key structure and support role in body, for instance, bone
and cartilage rely on the collagen for their strength. Other tissues such as nose and
outer ears consist largely on collagen.
• Collagen are form with a large number of Gly-Xaa-
Yaa tripeptide repeat, where Xaa and Yaa are usually
proline or 4-hydroxyproline.
• The collagen peptide chain interwind with two other
collagen chains to form a triple helix. This helix is
hold by hydrogen bonds formed between glycine N-
H to proline carbonyl in the other chain and so on.
• The 4-hydroxyproline is formed by post-translational
modification by enzyme, the stereoelectronic effect
from the hydroxyl group involves the helix stability.

stabilize destabilize
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CHEM5980

7 transmembrane domains
• Seven-transmembrane domains are
characterized by the presence of seven α helix
that traverse the cell membrane.
• The helices of the seven transmembrane are
common element for signal transduction. These
helices form a basket for binding small molecules,
which functions as signaling messengers.
• Using Rhodopsin as example, this G-protein
receptor bind aldehyde ligand retinal and form
imine with its lysine residue. Then light transform
the trans C-C double bond into cis and change the
conformation as a signal for the receptor to
transmit into cell through G-protein.
• α helix bundles are common as protein domains,
these helices usually have hydrophobic amino acid
on the location in contact with other helix to drive
protein folding in aqueous solutions.
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CHEM5980

Peptide-binding domains
• Sequence-selective peptide binding is often involved in protein communications.
SH2 and SH3 domains are examples toward this interaction.
• SH2 domain recognize a phosphate group on tyrosine, which is the result of a
signaling process. SH2 domain has antiparallel β sheet with a α helix at both top
and bottom.
• SH3 domains are β barrel and they recognize peptides with multiple prolines that
forms a helix structure, which has a three-fold symmetry viewing from the helical
axis.

SH2 SH3
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CHEM5980

Higher levels of protein structure

• Protein tertiary structure is defined by the folding of a individual peptide chain.
Usually such structure are composed by several domains.
• Tyrosine kinase Src is an example that the combination of multiple domains from a
single peptide chain to elicit protein function.
• Binding of kinase domain to C-terminal peptide with phosphotyrosine residue to
keep enzyme inactive. Src activator can be unleashed by tyrosine residue
dephosphorylation.
• https://www.youtube.com/watch?v=kbI-1Y63Sfw
 Section 5-6 National Tsing Hua University
CHEM5980

Higher levels of protein structure

• Quaternary structure consist of non-covalent bonded
proteins that assemble into large complexes.
• Ferritin, a iron storage protein, is an example of
quaternary structure that is composed of 24 identical
4-helix bundles.
• The assembly of collagen begins with C-terminal
domain fold into globular structure and trimerize to
nucleate triple-helix.
• Isomerization of proline is catalyzed by prolyl cis-trans
isomerase to help helix formation.
• Enzyme cleave globular domain to allow collagen helic
to form fibril.
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CHEM5980

Diseases arise from protein misfolding

• Protein folding is crucial to maintain their functions, misfolding of certain essential
protein cause diseases.

Nat Rev Mol Cell Biol 2014, 15, 384

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Disease Models & Mechanisms 2014, 9

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Mechanism of protein folding disease

Disease Models & Mechanisms 2014, 9

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CHEM5980

Mechanism of protein folding disease

Disease Models & Mechanisms 2014, 9

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CHEM5980

Mechanism of protein folding disease

AAT: α1-antitrypsin
Secreted protease inhibitor

Disease Models & Mechanisms 2014, 9

 Section 5-X National Tsing Hua University
CHEM5980

Mechanism of protein folding disease

Disease Models & Mechanisms 2014, 9

Section 5-X National Tsing Hua University
CHEM5980

Disease Models & Mechanisms 2014, 9

Section 5-X National Tsing Hua University
CHEM5980
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Alzheimer disease cause

• Alzheimer disease (AD) was first described in 1906.
Patients usually with age above 65.
• Cause for AD is largely unknown, only 1~5% are identified
to caused by generic difference. The competing
hypotheses include: Genetic heritage, cholinergic
hypothesis, amyloid hypothesis and Tau protein.
• From the identified generic caused cases, gene mutations
in 3 proteins are mostly found: amyloid precursor protein Image courtesy of the National Institute
(APP) and presenilins 1 and 2. on Aging/National Institutes of Health
• Cholinergic hypothesis proposes that the
reduced synthesis of the neurotransmitter
acetylcholine causes AD.
• Amyloid hypothesis proposed that
extracellular beta-amyloid (Aβ) deposits are
the fundamental cause of the disease.
• Tau protein is involved in the aggregates
formation as neurofibrillary tangle.
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Alzheimer disease pathology

• Alzheimer disease (Ad) can be characterized with the loss of neurons and synapses in
the cerebral cortex. Under microscope, both amyloid plaques and neurofibrillary
tangles are clearly visible.
• Amyloid plaque in AD is formed by the aggregation of a Amyloid-beta (Aβ), which is a
peptides of 36–43 amino acids. This peptide is form by proteolytic cleavage of
amyloid precursor protein (APP) by β- and γ-secretases.
• The monomeric Aβ rapidly forms disc-like oligomers with a dimension of 2nm in
height and 15nm in diameter, before it converts to fibril.
• The Aβ42 oligomer is
highly toxic to mouse
cortical neurons
compared to fibril.
Pentamer and hexamer
may be building block
for more toxic decamer
Aβ42 oligomer
and dodecamers.
• NMR structural studies
and hydrogen-
deuterium exchange
was applied for this
investigation. Aβ42 fibril Nat Struct Mol Biol 2010, 17, 561
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Alzheimer disease pathology

• Neurofibrillary tangle is a marker of AD.
• Tau protein is a microtubule-
associate protein that functions to
modulate the stability of axonal
microtubules.
• Tau protein can be phosphorylated
by kinase to disrupt microtubule
organization as part of regulation.
GSK3b
• However, when Tau protein is CDK5
hyperphosphorylated, it self- ERK2
assembly into tangles of paired
helical filaments and straight
filaments, which is observed as
neurofibrillary tangles.

Nat Rev Drug Disco 2007, 6, 464

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Alzheimer disease drugs

• Current approved Alzheimer's drugs do not cure or stop the disease from
progression, instead it reduces symptoms, such as memory loss and confusion, for
a limited time.
• Current AD drugs fall into 2 category:
1) Cholinesterase inhibitors work by slowing down the process that breaks down a
key neurotransmitter. Donepezil, galantamine belong to this group.
2) NMDA (N-methyl-D-aspartate) receptor antagonist works by regulating the
activity of glutamate, an important neurotransmitter in the brain involved in
learning and memory. Glutamate act on NMDA receptors to permit calcium to
enter the cell for signaling. In AD, however, excess glutamate can be released from
damaged cells, leading to chronic overexposure to calcium, which can speed up cell
damage. Memantine helps prevent this destructive process by partially blocking the
NMDA receptors.

Donepezil Galantamine Memantine

• Current direction for AD drug development focus on immunization strategy and

inhibitor for the enzyme that process Aβ precursor protein.
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Prion diseases
• Prions, derived from the words protein and infection, are responsible
for the several neuron diseases include bovine spongiform
encephalopathy (BSE, also known as "mad cow disease") in cattle,
scrapie in sheep, Creutzfeldt-Jakob Disease (CJD), and kuru in human.
• Prion protein (PrP) endogenous cellular glycoprotein that is expressed
predominant in the central nervous system.
• Prion protein contains a signaling peptide domain (1-22), polybasic region (23-27),
and His-rich region that binds Cu2+ and other metal ions. The hydrophobic domain
(111-130) is highly conserved and serve as a transmembrane anchor under certain
situation.
• During post-translational process in ER, signaling peptide is removed and GPI
anchor attached at residue 230, which allow most PrP to be found on cell surface.
• Recent studies suggest the functions of prion protein include: memory formation
and involved in copper homeostasis mechanism, calcium modulation, or a sensor
for copper or oxidative stress.
• Prion disease can arise in either sporadically as the result of prion protein mutation
or by infection from exogenous sources.

Trend Neurosci 2012, 35, 92

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Prion diseases
• Cellular prion protein in normal conformation is known as PrPc. A large number
of evidence suggest the key in the prion disease is the accumulation of scrapie
form of prion protein, PrPsc.
• In this process, the infectious PrPsc was used as a template to convert PrPc into
PrPsc in a catalytic manner.
• Although the infectivity of PrPsc has been confirmed, the toxicity, from which
species resulted in neural cell death is remain unclear.
• Evidences have indicated toxicity of PrPsc requires the presence of membrane –
anchored PrPc at the cell surface. So the normal activity of the protein is
involved to inducing toxicity.

Nat Rev Microbiol 2011, 9, 771

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Prion protein and Aβ

• Interestingly, recent
studies also suggest that
connections between two
neural disease: Alzheimer
and prion diseases.
• PrPc has been suggested
as a receptor for Aβ
oligomer, which was
thought as a more toxic
species than it amyloid
fibrils.
• The Aβ oligomer may just
act as PrPsc to membrane-
associated PrPc to deliver
a toxic signal to synapses.

Trend Neurosci 2012, 35, 92