Download Extrapolating Anfinsen`s conclusions…

MBLG1001 lecture5
page 1
What determines the conformation a protein will assume?
This question has vexed biochemists for some time. Because each protein has its own
unique fold the amino acid sequence was thought to have a major influence on the final
3D conformation the protein will assume. If the process was completely random and the
final fold was a process of trial and error it would take longer than the estimated age of
the universe to fold a polypeptide of 100 amino acids. That being the case correct folding
must be a more directed process.
One of the major series of experiments on protein folding was carried out by Anfinsen
using the protein ribonuclease. This protein is a small enzyme (124 residues) containing 4
disulphide bonds, which catalyses the breakdown of RNA. The enzyme can be treated
with 6 M guanidinium HCl and reducing agents which effectively denature the enzyme.
In this state it is unfolded, the S-S bonds are reduced and it has no activity. If this
preparation is then slowly dialysed to remove the guanidinium salts and the reducing
agent the activity slowly returns. After dialysis is complete 100% activity has returned.
The conclusion drawn from these results was that the information necessary to direct the
folding of the protein was contained in the primary amino acid sequence.
The role of disulfide bridges.
Disulfide bridges or bonds form between 2 cysteine residues in an oxidising environment.
Further investigation suggests that the formation of the disulfide bonds is not a random
event either. Ribonuclease has 4 S-S bonds, hence 8 cysteines. If the S-S bond formation
was completely random then the chances of forming the correct disulfide bonds is very
low. The chances of forming the first correct bond is 1 in 7, the second 1 in 5 etc. If the
process was completely random we would expect to achieve only a small fraction of the
original activity (~1%). The fact that 100% activity was achieved when sufficient time
was allowed indicates that correct disulfide bond formation is favoured when correct
folding takes place. Disulfide bonds are not essential for correct folding but they do
stabilise the protein once it is folded. Disulfide bonds are usually found in proteins which
are exported eg ribonuclease, insulin. This is probably because the exterior of the cell is
oxidising hence stabilises –S-S- formation while the interior of the cell has a reducing
environment, favouring the reduced –SH.
Extrapolating Anfinsen’s conclusions…
While these experiments are quite conclusive with a small protein in vitro the situation
inside the cell is different. The proteins are often much bigger than ribonuclease and the
environment has a higher protein concentration. The chances of misfolding are greater
and the possibility of misfolded proteins aggregating together is also greater. Misfolded
MBLG1001 lecture5
page 2
proteins will aggregate together if hydrophobic regions are inappropriately exposed. To
compound the problem further it is thought that misfolded proteins are responsible for a
number of disease states. To ensure this doesn’t happen chaperones are used. These
proteins make sure the protein folds correctly, speeding up slow parts of the folding
process and ensuring that inappropriate folding does not occur.
Diseases caused by incorrect folding cont….
Mad cow’s disease or bovine spongiform encephalitis (BSE) and a number of related
neurological disorders are now thought to be the result of protein misfolding. The
infectious agent is a prion and the protein responsible is a prion related protein or PrP.
Normally this PrP exists in the cell in a nonpathological form but under certain
circumstances it refolds to another conformation which causes enormous damage to the
nervous system. This abnormal form of PrP, if ingested, can cause the conversion of the
normal PrP in the recipient to the abnormal form, hence the disease is transmitted.
Why do proteins fold at all?
This question is not as silly as it sounds. Let’s go back to the laws of thermodynamics.
The second law states that everything moves to disorder (and they need a law to say
that!). More scientifically reactions proceed in the direction that will give molecules more
options. In the case of proteins wouldn’t the unfolded state have more options and be less
ordered than the folded state? By any stretch of the imagination the answer would be yes.
Yet proteins spontaneously fold in a reproducible manner to produce functional proteins.
Why or How??? To answer this question we must consider the entropy of the whole
system. While the protein may become more ordered ie entropy lowered the entropy of
the whole system, solvent molecules included will increase. This is because non-polar
side chains will become buried in the core of the protein and the water molecules that
were surrounding the side chains will be freer to assume more options.
Can functional proteins assume more than one conformation?
The answer here is also probably yes. Folded proteins are dynamic structures with a
number of slightly different functional conformations ie they are said to breathe.
Extra Information: Globular Proteins
Most proteins that have catalytic and regulatory roles are described as globular proteins.
They have a “spherical” shape and contain sections of more rigid alpha helix or beta
sheet interspersed with flexible random coiled regions. These folding patterns are well-
MBLG1001 lecture5
page 3
defined and consistent for the particular protein but “deformable”. The tertiary folding of
these proteins comes about from the folding of secondary structure elements, yielding the
final 3-D structure.
Folding in vivo
Globular proteins fold very rapidly inside the cell in a reducing environment. It would
seem that they begin to fold in several spots simultaneously. Once the process has started
these partially folded structures will funnel by energy minimisation to the final
“structures”. This whole process is all the more amazing given that the cellular
environment contains a high macromolecular concentration (~300mg/ml).
For larger proteins, chaperones are required; to protect the peptide during the folding
process when hydrophobic regions are exposed to solvent or to prevent inappropriate
association with other proteins. Some chaperones e.g. chaperonins bind to improperly
folded proteins via their exposed hydrophobic regions and then induce the protein to fold
correctly in an ATP driven process. This process is carried out inside a protective “cage”
which isolates the protein and prevents nonspecific aggregation to other unfolded
proteins. Other chaperones, known as heat shock proteins (hsp70) bind to the polypeptide
chain as it emerges from the ribosome and begin the folding process even before
polypeptide synthesis is completed. Another class, hsp90 proteins are involved in folding
proteins involved in signal transduction, in particular steroid receptors. The hsp90
proteins bind to steroid receptors and maintain their shape in the absence of steroid
hormones. (The steroid hormones enter the cell and bind to the receptors, releasing them
from the hsp proteins. The steroid receptor complexes then enter the nucleus and bind to
DNA enhancer regions, activating transcription of specific genes.)
DNA binding proteins.
These proteins can be broadly divided into 2 classes; non-base sequence specific
interactions and base sequence specific DNA binding.
Non-base sequence specific interactions:
There are a number of structural examples e.g. histones involved in the packaging of
DNA and enzymes e.g. DNA polymerases, ligases, topoisomerases and helicases.
Base sequence specific interactions:
MBLG1001 lecture5
page 4
There are a number of proteins which interact with the DNA in a base sequence specific
manner. This is no trivial achievement; remember the DNA is composed of bases and the
protein amino acids. Proteins cannot base pair to the DNA!!
The second problem is that the distinguishing features of DNA are buried in the middle
of the double helix, not exposed on the outside. DNA on the surface appears to be the
same, irrespective of the sequence. Somehow the protein side chains must ‘reach in’ to
the middle of the helix and ‘read’ the bases.
The main classes of proteins able to achieve this feat are certain polymerases that initiate
synthesis at a particular sequence, regulatory proteins such as repressors and transcription
factors and restriction endonucleases.
This will be revisited once we cover the structure of nucleic acids.
The common themes emerging from these DNA protein interactions are:
1. the base sequence of the DNA has a 2 fold symmetry, sometimes described as
palindromic.
2. the protein DNA interaction occurs on one side of the molecule
3. the protein has one or more alpha helices which fit neatly into the major groove of
the DNA, the major groove provides a window into the double helix
4. the protein is often a dimer to match the palindomic DNA sequence
5. specific side chains in the alpha helices interact with specific bases by h-bonding
e.g glu and lys are often involved
6. positively charges amino acids (lysine and arginine) interact with the DNA
phosphates to stabilize the base specific interactions.
To be able to ‘read’ the DNA base sequence the protein must interact with the h-bonding
groups on the base pair interface in the major groove; it is the only way to distinguish the
bases.
The DNA helix conformation does change subtly with different sequences. DNA binding
proteins use ‘indirect readout’ to detect specific sequences from the changes in backbone
conformation. Local changes in backbone conformation will facilitate or prevent binding
to backbone phosphates.
Structural Motifs in DNA binding Regulatory Proteins (for your
information only…not examinable)
Helix-turn-helix (HTH):
Two alpha helices separated by a sharp beta turn. The helix nearest the C terminal fits
neatly into the major groove. A number of the side chains in this helix interact with
specific bases. The other helix interacts with the DNA binding helix through hydrophobic
interactions and stabilizes the helix, locking it into the DNA. Common HTH proteins are
found in prokaryotes e.g. the trp repressor, the lac repressor and the CAP protein.
MBLG1001 lecture5
page 5
The zinc finger:
The zinc finger is a module, a eukaryotic DNA binding motif, consisting typically of 2
Cys residues separated by a pair of residues, then 12 residues followed by a His, 3
residues and another His. This (Cys-x2-Cys-x12-His-x3-His) motif can be repeated
between 2 and 60 times. A Zn2+ ion is coordinated between the 2 Cys and the 2 His side
chains and the 12 intervening residues loop around forming a ‘Zn finger’. This finger
interacts with ~5 nucleotides in the DNA through the major groove. Repeated fingers will
interact with successive major groove nucleotides. The Zn2+ ion stabilizes the structure
and keeps the alpha helix in place in the major groove. Zinc finger motifs are contained
in the steroid receptor superfamily of transcription factors.
The leucine zipper:
The leucine zipper is actually 2 polypeptide chains containing alpha helices with a ribbon
of leucine residues down one side. The hydrophobic residues interact, similar to the
coiled coil interactions in keratins. The Y shaped dimer with the coiled coil stem has
arms with a linked set of DNA contact surfaces. The coiled coil positions itself
perpendicular to the DNA helix on the minor groove. The arms then feed into the major
grooves from opposite sides of the DNA. The dimer may be a homo or hetero dimer
giving a greater variety of sequence specificity. Because the subunits do not have to be
identical the DNA base recognition sequence does not have to be palindromic. The
original example found was a set of liver transcription factors which specifically bind to
CCAAT sequences.
Enzymes Overview
Most reactions in living systems are catalysed, if not, they would proceed at a negligible
rate. These catalysts are called enzymes. As well as increasing the rates of
reactions enzymes can also be highly specific for their preferred substrate. Since the
reactions would be very slow in the absence of enzymes, control of reactions is normally
achieved by regulating the activity of the enzymes that catalyse them. Enzymes can be
localized in certain organelles and organized into pathways.
Context
The efficient functioning of your body requires certain pathways to be stimulated in
certain circumstances and to be down regulated at other times. The flight response to
danger, for example, very much depends on the ability of certain enzymes to be activated
dramatically (the key enzyme increases its activity 2 000-fold). After the danger has
passed the same enzymes must return to ground state. The way enzymes work and how
they are regulated is crucial to our understanding of all biochemical processes. Many
diseases are the result of inherited or acquired deficiencies in a particular enzyme.
Examples include porphyria, gout, phenylketonuria and many others. It is in this context
that we will investigate enzymes.
MBLG1001 lecture5
page 6
Enzyme Characteristics
Until a relatively short time ago all enzymes were thought to be proteins. A small group
of catalytic RNA molecules, termed ribozymes (one example of which is the ribosome),
have recently been characterised. This observation ie that not all the "doing" molecules in
cells are proteins once again raised the issue of the basis of early life. Could RNA-only
organisms have preceded the evolution of proteins and DNA? We shall not consider
ribozymes further.
Enzymes usually have molecular weights between 10,000 and 1,000,000 Da. Some
enzymes require no additional chemical groups other than their own amino acid residues
for catalytic activity. Some require and additional component known as a cofactor - these
may be simple metal ions, such as Fe2+, Mg2+, Mn2+, or complex organic or
organometallic molecules called a coenzyme. Most of the essential metal ions and
vitamins that we need in our diet are required because they act as cofactors in enzymes. A
deficiency of the vitamin or ion is tantamount to a deficiency of the enzyme for which it
acts as a cofactor.
Some enzymes require both a coenzyme and one or more metal ions for activity. A
coenzyme that is covalently bound to the protein is known as a prosthetic group. A
complete catalytically active enzyme together with its necessary cofactors is called a
holoenzyme. The protein part of such a complex (that is, excluding the cofactors) is
termed the apoenzyme. Holoenzymes often have many subunits. Some of these subunits
are essential for catalytic activity while others have a regulatory role. The essential
subunits form the core enzyme eg RNA polymerase, DNA polymerase.
The molecule which is acted upon by the enzyme is termed the substrate, and this is
converted by the enzymatic reaction into product(s). The enzyme is often named
"trivially" by adding the suffix "ase" to the name of the substrate. For example, urease
catalyses the hydrolysis of urea, and DNA polymerase catalyses the synthesis of DNA.
Other enzymes such as trypsin and chymotrypsin do not follow this nomenclature
scheme.
How do enzymes work?
Thermodynamics versus kinetics
Go back to the fundamental thermodynamics of a chemical reaction. Molecules all
possess an intrinsic amount of internal energy. This energy is based on the chemical
structure of the molecule; the atoms that make up the molecule and the bonds formed.
Each particular molecule has its own specific energy. This is measured at standard
conditions (25oC, atmospheric pressure 760 mm Hg and 1 mole of the compound). Some
of that energy is taken up with the wiggling and jiggling of the atoms in the molecule and
cannot be used to do useful work. The total internal energy of a mole of a compound can
be considered to be the enthalpy (denoted ∆Hf or ∆Ho). Once the effect of the wiggling
and jiggling is taken into account (denoted ∆S or entropy) we have the free energy
available to do useful work, ∆G. When considering a reaction the relationship between
MBLG1001 lecture5
page 7
the change in free energy of the products and the substrate (reactants), ∆G and the change
in enthalpy, ∆H is:
∆G = ∆H - T∆S (a)
When you consider that the wigglings and vibrations of the atoms is a temperature
dependent process and cannot be used to do anything useful the expression (1) makes
sense.
Now we can construct what is known as a reaction coordinate diagram, which plots the
free energy of the system against the progress of the reaction.
The thermodynamics: For a reaction to proceed spontaneously in the direction from
substrate (S) to product (P) the free energy change, ∆G (more properly ∆G0) must be
negative. A system is at equilibrium and there will be no net change if ∆G = 0. If ∆G
were positive the reaction would require an input of energy to proceed.
Equilibria and living systems.
A reaction at equilibrium is not desirable for living systems because there is no energy
available for useful work; ∆G is 0. In fact living systems only achieve equilibrium upon
death!
It is desirable to be as far away from equilibrium as possible. Then there will be more
available energy for useful work (∆G).
BUT if reactions all move to equilibrium how can living systems stay in a steady state
non-equilibrium position?
By being an open system and continually exchanging gases, food and energy with the
environment.
Let us consider the case where the uncatalysed reaction has a negative ∆G0 and will
therefore proceed spontaneously (termed exergonic). The term spontaneous is somewhat
ambiguous. Thermodynamically it means the reaction will proceed albeit very, very
slowly!!
The Kinetics of a reaction:
The overall ∆G0 for the reaction tells us nothing about the rate of the
reaction only the eventual equilibrium outcome. The rate of the reaction is
the kinetics.
A good example of this is the oxidation of glucose in the presence of molecular oxygen,
O2, to yield carbon dioxide (CO2) and water. The ∆G0 for this reaction is enormously
negative (-2870 kJ per mole of glucose) yet this reaction occurs at an imperceptibly slow
MBLG1001 lecture5
page 8
rate when glucose and oxygen are mixed. Do you observe your sugar at home
spontaneously combusting? Why? The answer lies in the activation energy (∆
∆G‡)
required to reach the transition state. This is a hill over which the reaction has to climb
on its way from substrate to product. The transition state is more properly defined as the
transient activated state of the substrate/enzyme complex in which the substrate is equally
likely to either reform substrate or form the product. The problem is that if ∆G‡ is very
large, then it is unlikely that the coming together of the reactants at normal temperature
and pressure will be sufficient to surmount the barrier.
If the temperature is raised most reactions proceed more quickly because the energies of
both the reactants and products are raised.
What enzymes don’t do!
o Enzymes don’t change the overall ∆G0 of the reaction. This is dependent on the
free energy of formation of the substrate(s) and product(s) and is the result of the
chemical nature/structure of the compounds themselves.
o Enzymes don’t change the equilibrium constant for the reaction.
o Enzymes don’t change the direction of the reaction i.e. if a reaction is endergonic
without an enzyme it will be with one.
Explanation: The ∆Go is related to the Keq
Definition: The ratio of the [product(s)] divided by [substrate(s)] at equilibrium i.e.when
∆G = 0 is the equilibrium constant ie. Keq = [product]/[substrate].
Standard state: 1 M [substrate(s)] and [product(s)], 25oC, pH 7; the conditions
assumed for ∆Go. ∆Go’ is the same conditions except at 37oC (often more relevant
biologically).
In fact the equilibrium constant Keq is related to the ∆G0 via the following relationship:
∆G0 = -RTlnKeq. (1)
∆G0 is the free energy obtained per mole, under standard conditions ie when the
[substrate] and [product] concentrations are 1 M. If you are not at standard conditions
then ∆G is related to ∆G0 by the following relationship:
∆G = ∆G0 + RTln[product]/[substrate]. (2)
Keq = [product]/[substrate] @ equilibrium. (3)
At equilibrium ∆G = 0, hence equation (1) is obtained. If the free energy of formation of
substrate and product molecules are inherently different then the only way to obtain a ∆G
of zero (ie equilibrium) is to change the relative concentrations of product and substrate.
MBLG1001 lecture5
page 9
Thus the relative amount of product and substrate adjust at equilibrium to ensure energy
balance ie. ∆G is zero. The ratio of the [product] divided by [substrate] when ∆G = 0 is
the equilibrium constant ie. Keq = [product]/[substrate].
Take the simple reaction S P. If the ∆G0 for this reaction is very negative this tells us
that the ∆Gf of the product is significantly less than that of the substrate (∆G0 being Σ∆Gf
product-Σ∆Gfsubstrate). For this reaction to achieve a ∆G of zero (equilibrium) there
would need to be a lot more product relative to the substrate, hence the reaction would
favour the formation of product and the Keq would reflect this e.g. the reaction catalysed
by the glycolytic enzyme pyruvate kinase has a ∆G0 of -23 kJ/mol and a Keq of 3.63 X
105. Clearly this reaction is exergonic and favours the formation of the product. It is
considered to be effectively irreversible. Another reaction in the same pathway has a ∆G0
of +1.1 kJ/mol and a Keq of 0.483. This is a freely reversible reaction.