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Protein Function
Proteins and Ligands
• Proteins work by interacting with other molecules. This
means that each protein must bind to the specific molecules it
interacts with.
– Any molecule a protein binds to is called a ligand for that protein.
Ligands can be small molecules or macromolecules, including other
proteins.
• The binding can be weak or strong: weak binding means only
brief interactions, while strong binding means long term
interactions, such as are found in large multi-subunit
structures like ribosomes
– The binding affinity of a protein for its ligand is measured
by the dissociation constant, KD.
Binding Sites
• Ligands bind to specific regions of the proteins called binding sites.
• Binding sites are usually cavities on the surface of a protein.
• The R groups of various amino acids lining the cavity interact with the
ligand by electrostatic forces, hydrogen bonds, and Van der Waals
interactions.
•
•
The amino acids involved in binding the ligand are very precisely placed, and they are
very conserved in evolution.
The other amino acids of the protein serve as a scaffold to help position the ligandbinding amino acids. They are often less conserved in evolution.
Aligned Protein Sequences
• These are the
NTPase
domains of 4
MutS proteins
from the
Plasmodium
malaria
parasite.
• Note: internal
gaps, ragged
ends, highly
conserved
regions, semiconserved
regions,
regions with
little
homology.
Antibody Binding Sites
• Antibodies are part of the
immune system. They are
proteins that bind tightly to
specific ligands.
– Antibody ligands are called
antigens.
•
The world produces vast
numbers of possible antigens so
the immune system must be
capable of producing billions of
different antibodies.
– Disease organisms often disguise
themselves by trying to have a
non-antigenic surface.
• The antigen binding sites are
formed by the variable regions of
both the heavy chain and the
light chain.
• Enzymes are proteins that catalyze chemical
reactions. Each cell contains thousands of different
enzymes. Each enzyme catalyzes a single reaction.
• The ligands for enzymes are called substrates, and
the enzyme converts a substrate molecule into a
product molecule.
• Enzymes are named for the reaction they catalyze:
DNA polymerase polymerizes DNA from
nucleotides, for example.
• There are several general mechanisms by which
enzymes work:
– Putting two substrate molecules into the precise alignment
needed for the reaction to occur
– Stablizing a transition state between suubstrate and
product
– Providing a microenvironment that helps the reaction. For
example, a highly acidic or a very reducing environment will
encourage many reactions
– Straining the conformation of a substrate to encourage it to
transition to the product.
Enzymes
Example Enzyme: Lysozyme
•
•
Lysozyme cuts the polysaccharide chains in the
peptidoglycan coat of bacteria. This causes the
bacterial cells to lyse due to osmotic pressure.
Lysozyme performs a hydrolysis reaction: it adds
water to a chemical bond to break it.
– One product gets the –H from the water, and the
other products gets the –OH.
• The lysozyme reaction is energetically
favorable: the free energy of the products is
less than the free energy of the substrate.
Thus, lysozyme just has to lower the
activation energy of the reaction to get it to
proceed.
•
•
•
•
Lysozyme lowers the activation energy by straining the
bond and by stabilizing the transition state (by allowing a
temporary covalent bond between the sugar and the
enzyme molecule).
Also, in the microenvironment on the reaction site, note
that glutamic acid is in the –COOH form and aspartic acid
is in the –COO- form. This implies a pH of about 4.0, quite
different from the pH of the cell, which is around 7.4.
To add water to the bond, the polysaccharide needs to
be in a distorted position, with its electron distribution
altered. This is called the transition state.
Getting into the transition state requires a lot of energy,
which is difficult to achieve just from random collisions
with water molecules. It is achieved by binding the
substrate to the enzyme and holding it there during the
reaction.
– The lysozyme molecule has a long groove on its
surface that holds 6 sugars in the peptidoglycan
polysaccharide molecule. The enzyme breaks the
bond between the 4th and 5th sugars.
Lysozyme
Lysozyme Action
• At the active site, the C-O-C bonds between the sugars is strained.
• A nearby glutamic acid (Glu35) in the –COOH form attacks the bond by
donating its H+ to the connecting oxygen atom. (ES in the diagram)
• This leaves the carbon from the upstream sugar (sugar D) with a strong
partial + charge. (ES)
• This carbon atom is then attacked by the –COO- from aspartic acid (Asp52),
forming a (temporary) covalent bond between the sugar and the enzyme.
(Transition State)
• The combination of the –COO- on Glu35 and the unstable sugar-enzyme
covalent bond splits a water molecule into H+ and OH-. The H+ goes to Glu35,
regenerating its uncharged carboxylic acid state (-COOH). The OH- gets
attached to the sugar carbon, leaving Glu35 in its original –COO- state.
• The two halves of the polysaccharide are then released, and the lysozyme
can catalyze another reaction. (EP)
Coenzymes
• Enzymes often use tightly bound small molecules, called
coenzymes, to perform the chemical reaction.
– Some coenzymes are metal ions: Zn, Ni, Co, Cu, etc.
– Other coenzymes are small organic molecules, some of which are
vitamins: thiamin, riboflavin, retinal, etc. Others, such as heme, are
synthesized from other molecules.
• Examples:
– Retinal changes its conformation when struck by a photon. The
photoreceptor protein rhodopsin is bound to retinal and detects the
changed conformation
– The iron atom in heme reversibly binds oxygen molecules
– Biotin transfers –COOH groups by forming a transient covalent bond
with the –COOH
– The zinc (Zn) atom in carboxypeptidase forms a covalent bond with
the peptide bond that carboxypeptidase cleaves.
Some Co-enzymes
Heme, with
and without O2
bound
Retinal: cis-trans change
after absorbing a photon
Biotin reversibly binds a
–COOH group, then
transfers it to another
molecule.
The Na-K Pump
• Many proteins perform important non-enzymatic
functions. One of these is membrane transport:
for most small molecules, going across the
membrane requires them to pass through a
protein channel. (this is a big subject in BIOS 303)
• Some molecules need to use ATP energy to
actively pump them up the concentration
gradient. The sodium-potassium pump (also
called Na-K ATPase) is a very important case: it
keeps the cell in a state of low sodium and high
potassium inside the cell, relative to the outside
environment.
• Each cycle of the Na-K pump pumps 3 Na+ ions
out and 2 K+ ions in.
– An important consequence is that the cell has a lower
internal potential than the outside: the interior is at
about -70 millivolts.
For most cells, the Na-K
pump uses about 20% of
all ATP. In neurons, it uses
up to 2/3 of the cell’s
energy.
How the Pump Works
• The pump forms a channel through the membrane.
• The pump has 2 conformational states: open to the interior of the cell, or
open to the exterior.
• When it is open to the interior, 3 sodium ions bind to sites in the channel.
– This just happens by diffusion—there is always lots of Na+ around.
• The pump binds to an ATP molecule and hydrolyzes it. This
phosphorylates an aspartic acid on the pump protein, and releases ADP.
• Phosphorylation of Asp changes the conformation of the pump, so it is
now open to the exterior.
• The 3 Na+ ions diffuse away, and 2 K+ ions bind to the channel.
• The binding of the K+ ions causes the phosphate group on the Asp to be
released.
• The unphosphorylated pump changes back to the original conformation:
open to the interior.
• The K+ ions diffuse away and the cycle starts again.
Motor Proteins
• Motor proteins can move in a directional manner.
Used for things like movement of the cell,
movement of chromosomes in cell division, muscle
movement, etc.
• The best studied motor protein system is myosin
and actin in muscle cells. This system also moves
materials from place to place within the cell, and
causes cells to move in an amoeba-like fashion.
• Actin is composed on 2 strands wrapped around
each other. The strands are composed of many
globular subunits.
– In non-muscle cells, the actin filaments are called
microfilaments, and they make up the cytoskeleton.
– Actin is found in all eukaryotic cells, and its amino acid
sequence is highly conserved
Stained microfilaments in
fibroblasts.
More Myosin
• Myosin is the actual motor protein:
it uses ATP energy to pull itself along
an actin fiber.
– Many different forms in the cell
– Composed of a head region that uses
ATP, a flexible neck, and a long tail that
interacts with other myosin molecules or
with cargo materials.
• Myosin in the muscle consists of 2
chains wrapped around each other,
with 2 head groups.
– Myosin filaments are composed of
several myosin molecules wrapped
up together.
– In muscle cells, the thick filaments
are composed of 2 groups of myosin
molecules joined tail-to-tail.
Myosin Action
• The myosin head group has 2 conformations: low energy and
high energy (cocked).
• In the high energy position, ADP and Pi (the products of ATP
hydrolysis) are bound to myosin, and the myosin head
attaches to an actin molecule. (1 on the next slide)
• The power stroke occurs when ADP and Pi are released: this
causes myosin to change configuration, pulling actin along
with it. (2)
• Then, a new ATP binds to the myosin head, causing it to
release the actin. (3)
• ATP hydrolysis occurs, returning the myosin head to its high
energy or cocked position. (4)
Myosin Action
Kinesin
• Kinesin is a protein that transports materials
down microtubule pathways, from the center of
the cell to the periphery.
• It uses ATP, and it seems to walk done the
microtubule, pulling its load behind.
• The exact mechanism isn’t clear, but seems to
involve winding and unwinding the twisted stalks.
• Great video:
https://www.youtube.com/watch?v=y-uuk4Pr2i8
Control of Protein Activity
• To keep its metabolism running efficiently, a cell needs to have the proper
amount of each enzyme. This requires control over the amount of enzyme
present and how active each enzyme is.
• Control at different levels:
– How much of a specific protein is made: transcription and translation
– How rapidly the protein is degraded
– Confining the protein to a specific compartment in the cell: e.g. lysosome,
mitochondria, etc.
– Regulation of protein activity by small molecules or covalent modification (our
current subject)
Allosteric Regulation
• Allosteric regulation occurs when a small molecule affects an enzyme’s
activity by binding to a site separate from the enzyme’s catalytic site and
changes the enzymes conformation.
– Many enzymes are controlled by small molecules that have different structures from their
substrates or products.
– The enzymes have 2 different binding sites: one for the substrate, and another for the
inhibitor.
– When the inhibitor binds to the enzyme, the enzyme changes its conformation, which makes
it less active.
Aspartate transcarbamoylase,
first step in pyrimidine
biosynthesis, is inhibited by
binding cytosine triphosphate
(CTP). The enzyme changes
its conformation, so the active
site is closed off.
Feedback Inhibition
• Feedback inhibition is a form of
allosteric inhibition that occurs when
the regulatory molecule is part of the
same biochemical pathway.
– The feedback inhibitor comes from a later
step in the biochemical pathway. If too
much inhibitor builds up, it slows the earlier
steps down.
– Complex biochemical pathways often have
several different feedback inhibition steps.
• Negative regulation: when an increase
in the regulatory molecule causes a
decrease in the enzyme’s activity.
• Positive regulation: when an increase
in the regulatory molecule causes an
increase in the enzyme’s activity.
Sample Biochemical Pathway
• Glycolysis and the
Krebs cycle, plus
some of the
pathways leading to
other chemical
compounds.
• Each step is
catalyzed by a
specific enzyme.
• The black words
and abbreviations
are compounds,
while the blue
abbreviations are
the enzymes.
Covalent Modification
•
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Protein conformation can be affected by
attaching various groups to the amino acid side
chains.
The most common example of covalent
modification is phosphorylation, where a
phosphate group is attached to the –OH group in
serine, threonine, or tyrosine.
Many complex events in the cell are regulated by
phosphorylation: for example, signaling
pathways that are activated by extracellular
signals and end by increasing transcription of
certain genes. Also, cell division.
Phosphorylation is a reversible reaction: a
protein kinase takes a phosphate group from
ATP and attaches it to the protein, and a protein
phosphatase removes the phosphate group
(dephosphorylation.
Depending on the system, phosphorylation can
either increase or decrease enzyme activity.
Cell Signalling Example
• When a specifc
growth factor (GF)
binds to a receptor
protein on the cell
surface, the
transcription factor
eIF-4G is activated
through a series of
steps.
• Many of the steps
involve activating
enzymes by
phosphorylating
them.
Other Covalent Modifications
• Other small molecules can be
attached to specific amino acids
on a protein. A few examples:
– Adding the fatty acid palmitate to
a cysteine causes anchors the
protein to the membrane.
– Adding the small polypeptide
ubiquitin marks a protein for
degradation.
– Adding acetyl groups to lysines on
histone proteins removes some of
their positive charges and allows
the chromatin to condense
together.
GTP-binding Proteins
•
•
Another major form of protein regulation comes from the binding of guanosine
triphosphate (GTP). Hydrolysis of GTP to GDP + Pi releases energy that is used to
change the conformation of the protein and do useful work. Proteins that use GTP in
this manner are called G-proteins.
Steps:
1.
2.
3.
4.
•
GTP binds to a site on the protein, activating it.
The protein slowly hydrolyzes the GTP to GDP + Pi. The protein becomes inactive.
GDP is released. Protein still inactive.
A new GTP binds to the protein.
Very common in the first step of signal transduction: the receptor protein interacts
with a GTP-binding protein. We will also see a lot of G-proteins in the processes of
transcription and translation.
GTP-binding Protein
Example
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EF-Tu is a protein involved in translating
messenger RNA into protein.
Specifically, EF-Tu detects proper base
pairing between the tRNA’s anticodon
and the codon of the messenger RNA.
If the tRNA’s anticodon matches the
messenger RNA codon, EF-Tu hydrolyzes
GTP to GDP and changes its
conformation.
This causes EF-Tu to release the tRNA
and the tRNA to become fully bound to
the ribosome.
EF-Tu thus serves a proofreading
function: it only releases the tRNA if
there is a match between the tRNA and
the messenger RNA codon.
EF-Tu in blue, GTP
in yellow, tRNA in
red
G-Protein Coupled Membrane Receptors
• External signaling molecules are detected
when they bind to a receptor protein
embedded in the cell membrane.
– The receptor protein changes its conformation,
which is detected inside the cell.
• Many receptors use a GTP-binding protein (Gprotein) to start the signal transduction
process.
– These G-protein coupled receptors all have 7
transmembrane alpha helices
– There are about 800 different G-protein coupled
receptors in the human genome.
• These G-proteins have 3 subunits, alpha (α),
beta (β), and gamma (γ). That is, the G-protein
is a heterotrimer. The alpha and gamma
subunits have covalently attached lipids that
keep them bound to the inner surface of the
membrane.
External surface of a G-protein
with its ligand (epinephrine)
bound.
G-Protein Activation
• The alpha subunit interacts with GTP.
• Normally, the G-protein is associated
with the receptor protein, has GDP
bound, and has all 3 subunits associated
together.
• When the membrane receptor binds to
its ligand (in this case, the hormone
epinephrine), GDP is released and GTP
binds.
• This leads to the dissociation of the
alpha subunit (with GTP) from beta and
gamma.
G-Protein Effects
• The activated alpha subunit (with bound
GTP), activates the enzyme adenyl cyclase,
which makes cyclic AMP (cAMP), a
“second messenger” inside the cell that
triggers the response of the cell to the
hormone.
– cAMP causes the activation of protein kinase A,
followed by a whole cascade of events, including
turning on some genes.
• The alpha subunit slowly hydrolyzes GTP
to GDP, which causes it to no longer
stimulate adenyl cyclase and allows it to
re-bind to the beta and gamma subunits
• In some systems, the beta-gamma
subunits, dissociated from alpha, activate
further steps in the signaling pathway.
Extra