Cell Biology & Molecular Biology
of The Cell
Lecturer
Dr. Kamal E. M. Elkahlout,
Assistant Professor of Biotechnnolgy
Lecture 2
Protein Structure and Function
Introduction to biotechnology registration for Biotech MSc
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Protein Central Dogma
• Proteins are large molecules that are formed as single, unbranched
chains of amino acid monomers
• – But, proteins can be turned into branched structures by ubiquitin
and other ubiquitin-like molecules
• There are 20 different amino acids commonly found in proteins
• • A protein’s amino acid sequence determines its three dimensional
structure (conformation)
• – Well, sort of….
• A protein’s 3-dimensional structure determines its chemical
function(s)
• – (along with a whole lot of different post-translational
modifications that can alter parts of its structure and change its
functions)
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All amino acids have the same
general structure
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Fig. 2-14: The 20 common
amino acids found in proteins.
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Basic amino acids have a positive
charge at pH 7.0
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Acidic amino acids have a negative
charge at pH 7
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4 of the hydrophilic amino acids are
polar, but uncharged
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The remaining amino acids have hydrophobic
and “special” functional groups
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Selenocysteine is the 21st
genetic encoded amino acid
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Amino acids are linked by an amide linkage, called a
peptide bond, to form polypeptide chains
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• Peptide bonds and the α carbon atoms form
the linear backbone of proteins, which is a
regular, repeating unit
• The functional groups of amino acids form
“side chains” that are connected to the
backbone.
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Polypeptide chains are flexible, but
conformationally restricted
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The shape of proteins is determined
through 4 levels of structure
• Primary: the linear sequence of amino acids
• Secondary: the localized organization of parts of
a polypeptide chain (e.g., the α helix or β sheet)
• Tertiary: the overall, three-dimensional
arrangement of the polypeptide chain
• Quaternary: the association of two or more
polypeptides into a multi-subunit complex
• The final, 3-dimensional, folded structure is
generally one in which the free energy of the
molecule is minimized
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Three types of weak, non-covalent bonds also constrain the
folding of proteins into their energy minimized 3-D structures
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Hydrophobic interactions also play
a role in determining protein shape
• Residues with hydrophobic side chains tend to
cluster in the interior of the protein molecule,
avoiding contact with water
• Polar side chains tend to be arranged on the
outsides of proteins in contact with the
aqueous medium
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All of these bonds are about 30-300 times
weaker than covalent bonds
• So why are they important?
• Many weak bonds applied together can
produce a large force.
• The stability of a protein is determined by the
combined strength of many non-covalent
bonds
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Secondary Structure
• The α-helix and the β-sheet are two regular
folding patterns found in almost all proteins
• What produces these structures and why are
they so common?
• They result from hydrogen bonding between
multiple N-H and C=O groups in the
backbone.
• Side chains are not involved in these
structures.
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The α-helical backbone is a rigid
cylinder with the amino acid side
chains protruding from its surface
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▲ FIGURE 3-2 Structure of a tripeptide. Peptide bonds (yellow) link the amide nitrogen
atom (blue) of one amino acid (aa) with the carbonyl carbon atom (gray) of an adjacent
one in the linear polymers known as peptides or polypeptides, depending on their length.
Proteins are polypeptides that have folded into a defined three-dimensional structure
(conformation).
The side chains, or R groups (green), extending from the carbon atoms (black) of the
amino acids composing a protein largely determine its properties. At physiological pH
values, the terminal amino and carboxyl groups are ionized.
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▲ FIGURE 3-3 The helix, a common
secondary structure in proteins. The
polypeptide backbone (red) is folded into a
spiral that is held in place by hydrogen bonds
between backbone oxygen and hydrogen
atoms.
The outer surface of the helix is covered by the
side-chain R groups (green).
Side chains protrude
from the surface of the
cylinder
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• α-helices can
form very
stable coiledcoil structures
through
hydrophobic
interactions
between nonpolar side
chains
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β-sheets are found in the core of
many proteins
β-sheets are rigid, relatively flat and
extended structures that are
stabilized by hydrogen bonds
between neighboring polypeptide
strands
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Secondary structure: the beta sheet
Where do the side chains go?
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β-sheets can be in either a parallel
or antiparallel orientation
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Most extracellular proteins are stabilized by covalent –S-S- cross
links Disulfide bond formation is catalyzed in the ER prior to export
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Protein domains represent another
important unit of organization
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Figure 3-12. A protein formed from four domains. In the Src (tyrosine kinase involved in signaling between
cells in multicellular animals) protein shown, two of the domains form a protein kinaseenzyme, while the
SH2 and SH3 domains (Src homolgy domain2 & 3) perform regulatory functions. (A) A ribbon model, with
ATP substrate in red. (B) A spacing-filling model, with ATP substrate in red. Note that the site that binds
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ATP is positioned at the interface of the two domains that form the kinase.
Hierarchical Structure of Proteins
• Domains are constructed from different
combinations of α-helices and β-sheets at
their core
• Each combination is called a protein fold
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• Most large multi-domain proteins have
evolved by recombination and joining of
preexisting domains in new combinations
(Domain Shuffling)
• Many small molecule binding sites in
proteins are created at the surfaces
between new combinations of domains
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Figure 3-18. Domain shuffling. An
extensive shuffling of blocks of
protein sequence (protein
domains) has occurred during
protein evolution. Those portions
of a protein denoted by the same
shape and color in this diagram are
evolutionarily related.
Serine proteases like chymotrypsin
are formed from two domains
(brown).
In the three other proteases
shown, which are highly regulated
and more specialized, these two
protease domains are connected
to one or more domains
homologous to domains found in
epidermal growth factor (EGF;
green), to a calcium-binding
protein (yellow), or to a "kringle“
domain (blue) that contains three
internal disulfide bridges.
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• Large proteins often contain more than one
polypeptide chain
• Binding between two protein surfaces
generally involves sets of non-covalent bonds
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•
•
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Figure 3-21. Two identical protein subunits binding together to form a symmetric
protein dimer. The Cro repressor protein from bacteriophage lambda binds to
DNA to turn off viral genes.
Its two identical subunits bind head-to-head, held together by a combination of
hydrophobic forces (blue) and a set of hydrogen bonds (yellow region).
Figure 3-22. A protein molecule containing multiple copies of a single
protein subunit. The enzyme neuraminidase (glycoside hydrolase nz,
neuraminin acid) exists as a ring of four identical polypeptide chains.
The
small diagram shows how the repeated use of the same binding
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interaction forms the structure.
• Figure 3-23. A protein formed
as a symmetric assembly of
two different subunits.
• Hemoglobin is an abundant
protein in red blood cells that
contains two copies of a
globin and two copies of b
globin.
• Each of these four
polypeptide chains contains a
heme molecule (red), which is
the site where oxygen (O2) is
bound.
• Thus, each molecule of
hemoglobin in the blood
carries four molecules of
oxygen.
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Some globular proteins
can form long helical
filaments
• Globular proteins fold into
a compact, ball-like shape
with irregular surfaces
• Example: Actin filaments
form in a helical
arrangement that can be
the length of the cell
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Proteins can be subunits for the
assembly of large structures
• enzyme complexes
• ribosomes
• Proteasomes (large proteases, degrade
uneeded damage proteins)
• filamentous structures (nuclear lamina)
• viruses
• membranes
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Protein Function
Some General Principles
• All proteins bind to other molecules
• Protein binding has a high degree of specificity
for its ligands (binding partners)
• Ligand specificity and affinity are determined
by sets of weak non-covalent bonds and
hydrophobic interactions.
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Figure 3-38. The binding site of a protein. (A) The folding of the polypeptide chain
typically creates a crevice or cavity on the protein surface. This crevice contains a set
of amino acid side chains disposed in such a way that they can make noncovalent
bonds only with certain ligands. (B) A close-up of an actual binding site showing the
hydrogen bonds and ionic interactions formed between a protein and its ligand (in this
example, cyclic AMP is the bound ligand).
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Enzymes are highly specific catalysts
• Enzymes speed reactions by selectively
stabilizing unstable transition states
(conformations) of their ligands
• This lowers the activation energy of the
reaction.
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The catalytic activities of many enzymes are highly
regulated through small molecule binding sites
• Allosteric enzymes have two or more binding
sites that interact with other molecules
• – an active site that recognizes substrates
• – a regulatory site that recognizes a regulatory
molecule binding of a regulatory molecule at
one site on the protein causes a
conformational change in the polypeptide that
can switch the active site conformation “On”
or “Off”.
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Figure 3-57. Positive regulation caused by conformational coupling
between two distant binding sites. In this example, both glucose and molecule
X bind best to the closed conformation of a protein with two domains. Because
both glucose and molecule X drive the protein toward its closed conformation,
each ligand helps the other to bind. Glucose and molecule X are therefore said
to bind cooperatively to the protein.
How does a cell regulate protein
function?
• Cells can regulate the steady state levels of proteins
through synthesis or degradation
• – Regulate mRNA levels by controlling transcription or
mRNA stability
• – Control of translation of a protein’s mRNA
• – Targeted degradation of a protein through
proteolysis
• Changing the activity of a protein through
conformational changes
• Changing the location of a protein by moving it to a
different part of the cell
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Protein structure and function can also be regulated
by covalent modifications of exposed residues
N-terminal acetylation stabilizes proteins
– non-acetylated proteins are degraded
rapidly by proteases
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Acetylation and Deacetylation of Histone
Tails Control Transcription Activity
• Deacetylation inhibits binding of transcription
factors to the TATA box, repressing gene
expression
• Hyperacetylation of histone N-terminal tails
facilitates access of general transcription
factors needed for transcription initiation
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A few of the chemical modifications found on
functional groups on internal residues
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Ubiquitin is one of a family of small proteins that can
be covalently linked to the ε- aminogroup of exposed
lysine residues
• Ubiquitin covalently links its C-terminal Glycine residue to
the ε-amino group of lysine through an iso-peptide bond.
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• Polyubiquitination
can target proteins
for degradation in
proteasomes
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Monoubiquitination, or Linkage of Small UbiquitinLike Molecules (SUMOS) Can Regulate Protein
Structure and Activity
• In cells, many changes in protein binding /
catalytic functions are driven by
phosphorylation
• Cells contain a large collection of protein
kinases and phosphorylases
• What amino acids are phosphorylated?
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Phosphorylated amino acids
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Protein phosphorylation and dephosphorylation play a major role in
regulating enzyme activity and in driving the regulated assembly and
disassembly of protein complexes
• Addition of a phosphate group (2 – charges) to
a residue can attract + charged side chains,
causing major conformation changes
• Attached PO4 groups can form structures that
can be recognized as binding sites by other
proteins.
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• Protein
phosphorylation is
reversible and can
act as a molecular
switch
• Dephosphorylation
can restore original
conformation and
activity of the
protein
Phosphorylation /
dephosphorylation
Note: In this case the dephosphorylated
form of the protein is active
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Other proteins bind and hydrolyze GTP to act as a
molecular switch (GTP binding proteins or GTPases)
• Actually another form of phosphorylation/
dephosphorylation
• GTP binds tightly to protein, usually activating
it.
• Protein can self-catalyze conversion from GTP
to GDP.
• Conformational change converts protein to
inactive form.
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Activation of Ras signaling causes cell
growth deferentiation and survival
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GEF guanine exchange facto, GAP GTPase activating factor
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Phosphoproteins can serve as signal integrators
for a molecular switch
• Example: Activation of a protein requires the input
of multiple signals from different parts of the cell
• cdk kinase (cyclin dependent kinase) – involved in
cell division
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Different graphical representations of
the same protein
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Different graphical representations of
the same protein
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