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Transcript
• There is a huge number of different threedimensional shapes possible, determined by
the amino acid sequence of the polypeptide.
• Function follows structure.
• There is an enormous versatility in protein
structure and therefore function.
Proteins are
made up of
amino acids
covalently
bonded together
by peptide
bonds.
alpha carbon
amino
terminus
carboxyl
terminus
Each protein has a unique sequence of amino acids. This
amino acid sequence specifies the shape of the protein due
to the fact that each protein folds into the most energetically
favorable conformation
Many of the covalent bonds allow free rotation of the atoms they join. so that the
polypeptide backbone can in principle fold up in an enormous number of ways.
However each chain, depending on the sequence of amino acids will be
constrained by many different sets of weak noncovalent bonds formed both by
atoms in the polypeptide backbone and the atoms in the amino acid side chains.
These weak bonds include hydrogen bonds, ionic
bonds, van der Waals attractions. A fourth weak force
important in protein folding is
hydrophobic/hydrophilic interactions
The distribution of polar and nonpolar amino acids is important
in how a protein folds. The nonpolar side chains tend to cluster
in the interior of a molecule, avoiding contact with water, while
the polar side chains arrange themselves near the outside.
Hydrophobic areas also tend to be found spanning the
lipid bilayer of membranes like the plasma membrane.
enzyme lysozyme
Large numbers of
hydrogen bonds form
between adjacent
regions of the
polypeptide chain and
help stabilize its
three-dimensional
shape.
Each protein normally folds into a single stable conformation – of
lowest energy. This conformation will change slightly during
interactions with other molecules (as in enzyme-substrate
complexes). This change in shape is often crucial for the function
of the protein. Ex. receptor proteins
See question 5-1
This conformation (the 3-D shape) is specified by its amino
acid (aa) sequence. The non-covalent bonds and
hydrophobic/philic interactions which hold a protein in the most
energetically favorable conformation depend entirely on the aa
sequence.
Molecular chaperones assists protein folding
and prevent newly synthesized protein chains
from associating with the wrong partners. Make
protein folding more reliable.
However, all the information required
for proper protein folding is contained in its
amino acid sequence.
Proteins in a cell are found in a range of sizes.
Protein sequencing has been replaced by DNA
sequencing, which is much easier.
Three-dimensional structure is determined by xray crystallography and NMR specroscopy.
Panel 5-6, pg. 165.
C-terminus
phosphocarrier
protein HPr, a
transport proteins
that facilitates sugar
transport into
bacterial cells
Nterminus
polypeptide
backbone model
ribbon model
wire model
includes amino
acid side chains
space-filling
model
Find two regular folding patterns: alpha helix and beta sheets.
Both result from hydrogen-bonding between the N-H and C=O
groups in polypeptide backbone, without involving side chains
Every 4th
peptide
bond
Complete
turn every
3..6 aa
1/2 bonds
with 4-5
Alpha helix is found in alpha-keratin, abundant in skin, hair, nails
The polypeptide chains are held together by hydrogen bonds between peptide
bonds in different strands. The amino acid side chains in each strand
alternately project above and below the plane of the sheet.
Antiparallel
Parallel
Coiled-coil
Ex. Alpha-keratin,
forms intracellulr
fibers that reinforce
the outer layer of the
skin
And
myosin
molecules
in muscle
cells
Hydrophobic helices also tend to be found spanning the
lipid bilayer of membranes like the plasma membrane.
Channel proteins often have hydrophobic exteriors and
hydrophilic interiors.
Domains are produced by any part of a
polypeptide chain that can fold independently
into a compact, stable structure - a modular
unit. Proteins often have more than one
domain - each with a specific
function.
Binds
DNA
Binds cyclic
AMP
(intracellular
signaling
molecule)
Turns genes on or
off
Catabolite activator protein (CAP)
• CAP is a bacterial signal transduction molecule
• The large domain binds cyclic AMP, an
intracellular signaling molecule. When cyclic
AMP binds it causes a conformational change
in the protein that enables the small domain to
bind to a specific DNA sequence and turn on
adjacent genes.
Cytochrome b, a
single-domain
protein involved in
electron transfer in
E. coli
NAD-binding
domain of the
enzyme lactic
dehydrogenase
The variable
domain of the
immunoglobulin
(antibody) light
chain - a beta
barrel
Notice loops at each turn
• The polypeptide chain generally passes
back and forth across the entire domain,
making sharp turns only at the protein
surface. The protruding loop regions often
form the binding sites for other molecules.
• For each protein, a single conformation is
extremely stable and has the exact chemical
properties that enable the protein to perform a
particular catalytic or structural function.
• Proteins are so precisely built that the change of
even a few atoms in one amino acid can
sometimes disrupt the structure and the function of
a protein.
• Proteins can be grouped into families with very
similar sequences and structures, probably due to
genes duplicating and evolving.
Serine protease family: elastase, trypsin, chymotrypsin, and some
proteases in blood clotting.
Green portion: aa sequence is the same
Notice the structural similarity and active site in red.
Each cleaves
between
peptides
different proteins or the bonds
different
Serine
Larger protein molecules may contain more than one
polypeptide chain or subunit. The region that interacts with
another molecule through
noncovalent bonds is the
binding site.
Hemoglbin contains two alpha globin subunits and two
beta globin subunits.
Heme is the
site where
oxygen is
carried
There are
many
large
multisubunit
proteins in
cells.
Globular proteins
Proteins with one
binding site can
form a dimer
Proteins with two
different binding
sites will often
form a long
helical filament
or a closed ring
An actin filament, a helical array of actin proteins
which can extent for micrometers in a cell thousands of actin molecules
Many large structures such as viruses and ribosomes
are built from a mixture of different proteins plus RNA
or DNA molecules. These structures can be isolated,
dissociated, and often spontaneously reassemble into
the original structure. Much of the structure of a cell is
self-organizing.
Aminoacids - alpha helix
Actin molecules actin filaments
Can be right or left handed
(screw=right (clockwise)
same when turned
upsidedown
Globular proteins
fold up into compact
shapes, like irregular
ball. Most enzymes,
even large and
complex enzymes are
globular.
Fibrous proteins are
elongated.
Coiled-coil
Ex. Alpha-keratin, a
dimer, forms
intracellular fibers
(cytoskeleton) that
reinforce the outer
layer of the skin
Capped by globular domains
which are binding sites,
allowing assembly into
ropelike, stable, intermediate
filaments in skin, hair,horns.
Outside of the cell, fibrous proteins form the gel-like
extracellular matrix. Secreted by cells, they assemble into
sheets or long fibrils.
Collagen, the most abundant in animal
tissues, consists of three long polypeptide
chains, each with the nonpolar aa glycine at
every third position. Wind around each other
in long regular triple helix and bind to one
another side-by-side and end-to-end
Elastin is formed from relatively
loose polypeptide chains which are
covalently cross-liked into a
stretchy meshwork
Skin, arteries
lungs
Hold tissues together.
Extracellular proteins are often stabilized by covalent cross-linkages. Esp
disulfide bonds. These form as proteins are being exported from cells,
catalyzed in the er by a special enzyme. Disulfide bonds do not typically
form in the cell cytosol. They also do not change the protein’s
conformation, but stabilize -reinforce - it.
The binding of a protein to another protein (its ligand) is highly
selective. Many weak noncovalent bonds – hydrogen bonds, ionic
bonds, van der Waals attractions - plus favorable hydrophobic
interactions are needed. Therefore the ligand must fit precisely
into the protein’s binding site.
The strength of the ligand/protein binding
depends on the strength and number of covalent
bonds and determines how long these molecules
will stay together. Random movements due to
thermal energy are always taking place.
The folding of the polypeptide chain typically creates a crevice or
cavity on the protein surface. The amino acids involved in the
binding site are often widely separated regions of the polypeptide
chain brought together when the protein folds. These amino acids
make many noncovalent bonds with the ligand
cyclic AMP
Other areas on the protein may contain binding sites for other ligand,
some of which may regulate the proteins activity or place the protein in
a particular location..
• Amino acids not in binding sites are usually
important for the general shape of the protein,
essential scaffold that gives the surface its
contours and chemical properties.
• These areas are often the secondary structures and
domains of the protein, that give it its 3dimensional shape – beta-sheets and alpha-helices.
• Therefore, mistakes in the amino acids in these
domains can change the 3-dimensional shape and
destroy its ability to function
• Strong binding is required when molecules must
remain tightly bound for long periods of time – for
example, ribosomes or proteosomes
Antibodies (immunoglobuilins)
are proteins made by our
immune system that can bind
virtually any molecule,
including those on
microorganisms. Each binds a
target molecule (antigen) very
tightly and with remarkable
specificity.
beta-barrels
The amino acid sequence in the loops is
hypervariable, the DNA is actually
changed in each one. The remainder of
the domains are structural and contain
binding sites for receptors on
phagocytes.
As ligands diffuse and bind to the antibody (or any protein binding site) and more and more
antigen-antibody complexes take form, the reverse reaction will begin to take place. When
association and dissociation take place at the same rate (free energy change = 0) the
equilibrium constant will be
The equilibrium constant is a measure of the binding strength.
The larger the equilibrium constant the tighter the binding
between protein and ligand.
• Enzymes not only bind to specific ligands,
called substrates, they also covert them into
chemically modified products.
• The enzymes remain unchanged.
• They speed up reactions, often by a factor
of a million or more.
• Can be grouped into function classes. Table
5-2.
• Each enzyme is specific for a single type of
reaction.
Lysozyme is an antibiotic found in egg white, saliva, tears, and
other secretions. It catalyzes the cutting of polysaccharide chains
(hydrolysis) in the cell walls of bacteria, which results in their
lysis.
The structure of lysozyme was worked by x-ray
crystallography. It has a binding site (active site)
that precisely fits the substrate. This is the site of
the chemical reaction. The active site holds 6
linked sugars at the same time in an transition
state – the atoms in the substrate are held in a
slightly altered geometry. The enzyme quickly
hydrolyzes the bond and releases the substrate
Conditions are created in the microenvironment of the lysozyme active site that greatly
reduce the activation energy needed.
glutamic acid
high conc of H+
aspartic acid
D. Some enzymes briefly form a covalent bond
between the substrate and a side chain of the enzyme.
This bond is broken in the end, leaving the enzyme
unchanged. Figure 4-5.
As the concentration of the substrate increases, the rate of the
reaction increases in a linear fashion. As the enzymes active
sites become saturated with substrate the rate increases only
slightly until the maximum value is reached – Vmax.
The rate of product formation now depends only on the speed
of the enzyme. This turnover number is often around 1000
substrate molecules per second!
The concentration of substrate needed to allow efficient an
enzyme rate is κM at which the enzyme works at half its max
speed.
Tightly bound small molecules add functions to
proteins.
rhodopsin = the purple light-sensitive pigment
made by the rod cells in the retina which detects
light by a small molecule retinal, embedded in
the protein. Retinal changes its shape when it
absorbs a photon of light and transmits this
shape change to the protein, which triggers a
cascade of enzymatic reactions that lead to an
electrical signal carried to the brain.
Hemoglobin
contains a heme
group which is
tightly bound to the
protein.
• Enzymes often have non-protein small molecules
as essential components.
– retinal and heme
• Sometimes these small molecules are covalently
attached
– cell membrane proteins covalently attached to lipid
molecules - lipoproteins
– glycoproteins, cell membrane or secreted.
• Enzymes often have a small molecule or metal
atom tightly associated with their active site that
assists in the catalytic function.
– carboxypeptidase – zinc
– biotin transfers a carboxylate group (a vitamin)
The metabolic system in the cell is so complex that elaborate controls are required
to regulate when and how rapidly each reaction occurs. Regulation occurs at
many levels. Transcriptional, Translational, Confining an enzyme to a particular
space, enclosed in or a part of a membrane (mitochondria). Most rapid and
general process is at the level of the enzyme itself.
1. Feedback inhibition
product
inhibits one
of the first
enzymes in
the pathway
multiple points of control
Positive regulation
occurs when a product
in one branch
stimulates activity of
an enzyme in another
pathway.
Ex. accumulation of
ADP activates several
enzymes involved in
oxidation of sugar
molecules.
Allosteric regulation – the regulatory molecule binds to another
binding site on the enzyme, rather than at the active site. The
binding of the regulatory molecule changes the shape of the
enzyme and the active site changes shape. In negative regulation,
it is longer able to bind to its substrate.
induces a conformation change
This mechanism (conformation change) is important in the function of other proteins
also. Receptors, structural proteins, motor proteins.
Conformational change in positive feedback.
2.
A second regulatory method is to add a
phosphate group covalently to one of the
amino acid side chains of the enzyme
(serine, threonine, or tyrosine). This
causes a major conformational change
which changes the activity of the
enzyme.
Reversible protein phosphorylation
controls the activity of more than a third
of the proteins in a typical mammalian
cell. It occurs as a response to signals
received from hormones, and
neurotransmitters.
Protein kinases phosphorylate enzymes
and protein phosphatases
dephosphorylate them.
3. GTP-binding proteins form molecular switches in response
to a signal received by the cell.
are usually active when GTP
often bind to other proteins to control enzyme
activities
crucial role in intracellular signaling pathways
Bacterial elongation factor EF-Tu
a GTP-binding protein
Allosteric transition = shape
change
A small change is magnified by
conformational changes within the
protein to produce a much larger
movement.
• Enzymes are regulated by negative feedback
inhibition (products inhibit an early enzyme) Fig.
32 and 33
– Often by allosteric interactions in which binding of a
molecule at one site changes its shape at a different
binding site
• Conformation (allosteric) change can be driven by
protein phosphorylation (Fig. 36)
• Also by binding GTP (GTP-binding proteins) Fig
37 and 38
• Enzymes can also be activated by proteases which
cleave off a segment – changing the shape of the
enzyme.
Motor proteins
- muscle contraction,
intracellular movement of
organelles, chromosomes,
enzymes along DNA, etc.
Without energy output, these
movements will be random –
back and forth
Not useful
Requiring energy output
makes these series of
conformational changes
essentially irreversible.
Unidirectional
An orderly transition
among three
conformations driven by
the hydrolysis of a bound
ATP molecule.
Would
require
ADP 
ATP
Hydrolysis of ATP or GTP drives an ordered series of
conformation changes. These changes effect each
polypeptide chain.
Proteins often form large complexes
that function as protein machines
EX. DNA replication, protein
synthesis, vesicle budding,
transmembrane signaling