Download 6. protein folding

Protein Folding
PROTEIN FOLDING
• Process in which a polypeptide chain goes from a
linear chain of amino acids with vast number of more or
less random conformations in solution to the native,
folded tertiary (and for multichain proteins, quaternary)
structure
Protein Folding
• Protein folding considers the question of how
the process of protein folding occurs, i. e.
unfolded  native state.
• This very challenging problem has been
described as the second half of the genetic
code, and as the three-dimensional code, as
opposed to the one-dimensional code
involved in nucleotide/amino acid sequence.
• Importance:
– Predict 3D structure from primary
sequence
– Avoid misfolding related to human diseases
– Design proteins with novel functions
Why do proteins fold?
In order to carry out their
function (for instance as
enzymes or antibodies),
proteins must take on a
particular shape, also
known as a "fold." Thus,
proteins are truly amazing
machines: before they do
their work, they assemble
themselves! This selfassembly
is
called
"folding."
Forms determines function
Suppose you have some molten iron. You may
turn it into nails, hammers, wrenches, etc. What
makes these tools different from each other is
their form (i.e. their shape and structure).
PRIMARY STRUCTURE DETERMINES TERTIARY (AND
QUATERNARY) STRUCTURES.
– demonstrated by the fact that many proteins can
refold from a more or less "random coil" set of
conformations without "instructions" from any other
cellular components
– All the information for 3-dimensional structure is
provided by the amino acid sequence.
Proteins fold on a defined pathway (or a small
number of alternative pathways); they don't
randomly search all possible conformations until
they arrive at the most stable (lowest free energy)
structure.
Anfinsen Experiment
•Denaturation of
ribonuclease A (4 disulfide
bonds), with 8 M Urea
containing bmercaptoethanol, leads to
random coil and no activity
Anfinsen Experiment
• After renaturation, the refolded protein has native
activity, despite 105 ways to renature the protein.
• Conclusion: All the information necessary for
folding into its native structure is contained in the
amino acid sequence of the protein.
Refolding
How do proteins fold?
Proteins have primary structures, which is their sequence
of amino acids, and secondary structures, which is the
three dimensional shape that one or more stretches of
amino acids take. The most common shapes are the alpha
helix and the beta conformation.
Proteins fold, amazingly quickly: some as fast as a
millionth of a second (microsecond)
• Folding means arriving at the right combinations of angles for every
amino acid residue in the sequence.
• Proteins fold on a defined pathway (or a small number of alternative
pathways); they don't randomly search all possible conformations until
they arrive at the most stable (lowest free energy) structure.
• Proteins that don't (re)fold on their own, need "molecular
chaperones" (which are also proteins) to keep them from slipping off
the folding pathway or to help them to get back on it.
–Some chaperones require ATP to carry out their function.
Interactions between the side chains of amino acids
determine how a long polypeptide chain folds into
the intricate three-dimensional shape of the
functional protein. As a peptide folds, its amino
acid side chains are attracted and repulsed
according to their chemical properties. For
example, positively and negatively charged side
chains attract each other. Conversely, similarly
charged side chains repel each other. In addition,
interactions involving hydrogen bonds,
hydrophobic interactions, and disulfide bonds all
exert an influence on the folding process. This
process of trial and error tests many, but not all,
possible configurations, seeking a compromise in
which attractions outweigh repulsions. This
results in a correctly folded protein with a lowenergy state
CHAPERONES
It is generally accepted that the information needed for
correct protein folding is contained in the primary
structure of the polypeptide. Given that premise, it is
difficult to explain why most proteins when denatured
do not resume their native conformations under
favorable environmental conditions. One answer to
this problem is that a protein begins to fold in stages
during its synthesis, rather than waiting for synthesis
of the entire chain to be totally completed. This limits
competing folding configurations made available by
longer stretches of nascent peptide.
In addition, a specialized group of proteins, named
“chaperones,” are required for the proper folding of
many species of proteins. The chaperones—also
known as “heat shock” proteins—interact with the
polypeptide at various stages during the folding
process. They help guide the folding and can help
keep the new protein from associating with the wrong
partner
Some chaperones are important in keeping the protein
unfolded until its synthesis is finished, or act as
catalysts by increasing the rates of the final stages in
the folding process.
Others protect proteins as they fold so that their
vulnerable, exposed regions do not become tangled in
unproductive interactions
HSPs in protein folding
•The diagram shows the role
of heat-shock proteins and a
chaperonin in protein
folding. As the ribosome
moves along the molecule of
messenger RNA, a chain of
amino acids is built up to form
a new protein molecule. The
chain is protected against
unwanted interactions with
other cytoplasmic molecules
by heat-shock proteins and a
chaperonin molecule until it
has successfully completed
its folding.
One major function of chaperones is to prevent both
newly synthesised polypeptide chains and assembled
subunits from aggregating into nonfunctional structures. It
is for this reason that many chaperones, but by no means
all, are heat-shock proteins because the tendency to
aggregate increases as proteins are denatured by stress.
Many chaperones are heat shock proteins, that is,
proteins expressed in response to elevated temperatures
or other cellular stresses. The reason for this behaviour is
that protein folding is severely affected by heat and,
therefore, some chaperones act to prevent or correct
damage caused by misfolding.
Other chaperones are involved in folding newly made
proteins as they are extruded from the ribosome.
Although most newly synthesized proteins can fold in
absence of chaperones, a minority strictly requires them
for the same.
Protein denaturation
Hydrolysis of Peptides and Proteins
• Peptide bonds are amide bonds and are resistant to
hydrolysis
• However, they can be hydrolyzed with enzymes or with
strong acid or base and heat
• Proteins are hydrolyzed in the stomach with both acid (HCl)
and enzymes (such as pepsin)
- the amino acids are then absorbed in the intestines and used
to synthesize new proteins
• Below is the acid hydrolysis of the dipeptide Ala-Ser to form
the amino acids alanine and serine
OH
CH3 O
+
H3N CH C N
heat,
+
H2O, H
CH2 O
CH C OH
OH
H
+
H3N
CH3 O
CH COH
CH2 O
+
+ H3N CH C OH
Denaturation of Proteins
• Denaturation causes proteins to lose their 3-D structure
and so they lose their function
• Denaturation involves the disruption of cross-linking in the
secondary, tertiary and quaternary protein structures
• Heat and organic compounds disrupt H-bonding and
hydrophobic interactions
• Acids and bases disrupt H-bonding between polar R
groups and break ionic bonds
• Heavy metal ions break S-S bonds by reacting with the
sulfur
• Agitation such as whipping stretches chains, disrupting all
types of cross-linking
Applications of Denaturation
• Denaturation of protein occurs when:
- an egg is cooked
- the skin is wiped with alcohol
- heat is used to cauterize blood vessels
- instruments are sterilized in autoclaves