Download Review the mechanism of protein folding

Review the mechanism of protein folding
SCOPE OF PROTEIN FOLDING
Proteins are the bio molecules which play pivotal role in this living world. They
are responsible for expression of certain characters in different types of cells
and constitute around 50% of the total cell dried mass. Proteins are the chain
of amino acids which binds with polypeptide backbone and then fold in a
unique 3D (native) structure by which protein expression takes place. Various
forces and factors are responsible for protein folding .If right expression of the
protein will not take place, it will cause disorders in human body. Many
diseases like Alzheimer’s; Parkinson, cystic fibrosis etc (Baldwin 2007) is
caused due to improper folding of proteins. For proper expression of protein,
the amino acid chain should be in its unique 3D structure. Sometimes
proteins require assistance in folding, molecules which help in the folding are
known as chaperones. These molecules help in the folding of the certain
protein molecules and also prevent the unfolding of the molecules.
In this assignment, I have tried to describe the mechanism of the protein
folding and effect of various factors which influence protein folding by taking
into consideration the present developments in our understanding of
thermodynamics and kinetics of protein.
INTRODUCTION
Protein folding refers to the process by which a protein assumes its
characteristic structure, known as the native state. Protein folding is very
complex mechanism and great development in its understanding has been
achieved in last 20years due to the development and use of some
sophisticated modern techniques like X Ray Crystallography, N.M.R, and
Mass Spectrometry etc. In the last few decades, we have managed to find the
3D structure of various proteins and how actually protein folding takes place.
In the complex process of protein folding, various factors act together to
construct specific 3D structure of a protein. Protein folding is a very quick
process taking milliseconds to seconds.
Due to high speed of folding, it is not possible to find each and every possible
conformation in fractions of time. Levinthal stated that each protein can
possibly have millions of pathway by which a desired 3D pattern can be
achieved. By study of intermediates we can understand the mechanism or
pattern on which protein folding works. But it is impossible to find out all
possible structure of protein molecules.
Protein folding study uses denaturants which help to denature the protein.
The logic is to use denaturing conditions viz. high pH, Temp, Pressure etc to
stop folding at intermediate stages (Stop flow technique) to study these
intermediates, thereby generating an overview of the whole process. Fully
folded structure is also known as native structure. Protein folding studies
have also been done in in-vivo and in-vitro conditions. In in- vivo protein
folding, some metal ions act as cofactors. These cofactors stabilise and
accelerate protein folding and finally help to achieve native stage
.Macromolecules like chaperones initiate protein folding, helping the protein
molecule to retain its folded confirmation and making it stable. In-vitro
concentration of macromolecules is very less, around 1% of what is originally
present in the cell. So inside cell, these molecules also affect protein stability
and make them more stable than in vitro (Rumfeldt et al. 2008)
Protein folding can be a two stages or multistage process, depending upon
the type of sequence. A single point mutation can change folding from two
stages to multistage and vice-versa .Some previous studies show that protein
sequences of less than 80 amino acids prefer two stage folding and large
protein sequences having more than 130 amino acids prefer multi stage
folding. Later studies have shown that sequences rich in F and G amino acids
prefer two stages folding where as sequences rich in C, H, L and R amino
acids prefer multistage folding (Ma, Chen & Zhang 2007)
PROTEINS FOLDING
Hydrophobic and hydrophilic interaction
Proteins are made of amino acids and joined by polypeptide bonds leads to
formation of polypeptide backbone. We have only 20 type of the amino acids
.whose combinations code for such complex structure and folding .These
amino acids can be divide in two groups one is hydrophilic or polar group and
other is hydrophobic or nonpolar group and their interaction with the
cytoplasm make them folded(Trevino, Scholtz & Pace 2007)
POLAR OR HYDROPHILIC AMINO ACID
NON-POLAR OR HYDROPHOBIC AMINO ACID
ASPARTIC ACID ”D”
ALANINE “A”
GLUTAMIC ACID ”E”
GLYCINE ”G”
ARGNIN ”R”
VALINE ”V”
LYSINE ”K”
LEUCINE ”L”
HISTIDINE ”H”
ISOLEUCINE ”I”
ASPARAGINE ”N”
PROLINE ”P”
GLUTAMINE ”Q”
PHENYLALANINE ”F”
SERINE ”S”
METHIONINE ”M”
THREONINE ”T”
TRYPTOPHAN ”W”
TYROSENE ”Y”
CYSTEINE ”C”
In the early stages of research, problem was why protein folds to a specific
structure and which part of the protein carried information for the folding and
later on this was find that primary structure of the protein code for the final 3D
structure. Hydrophilic and hydrophobic interactions of the amino acids adjust
protein it such a way that it suffers minimum repulsion and problem from the
surrounding. In folded state of protein all hydrophilic amino acid molecules
come at the other side and interact with water in the cytoplasm and
hydrophobic molecule come at the inner side of the molecule and don’t show
any reaction and attraction with water molecules and ultimately protein folding
take place in such a way that suffer minimum repulsion (Baldwin 2007)
All amino acids have different energetic in secondary structure. There are
many chameleon sequences in protein structure which can be taken as alpha
helix or beta sheets depending upon the tertiary structure of protein. These
chameleon are stabilised by hydrophobic forces (Chen et al. 2008)
Fig.a shows yellow colour hydrophobic molecules inner side of structure
Fig.b shows folding takes place and keeps hydrophobic molecules in inner
side
(Chen et al. 2008)
Apart from these interactions hydrogen bonding is very important in the
protein folding.
“Hydrogen bonding takes place between hydrogen
and electronegative atoms”
In the maintenance of the native structure hydrogen bonding present in
between the polypeptide amino acid chain which helpful in the formation of
the secondary, tertiary and quaternary structure of the proteins. Hydrogen
bonding also interacts between polar and side chain residues with the
surrounding water molecules. During the denaturing of the protein hydrogen
bond between protein molecules break and native structure of protein
disturbed (Djikaev, Ruckenstein 2010) Hydrophobic interactions are also
responsible for the protein folding. During hydrophobic interactions amino
acids which are non polar or hydrophobic they align themselves in such a
way that all hydrophobic come together and all hydrophilic molecules make
hydrogen bonds with water molecules, all hydrophobic amino acid come in to
inner side of the protein molecules and formation of the nuclei take place
which is hydrophobic these interaction further helpful in the secondary,
tertiary structure and due to this protein stabilise and help to achieve native
structure because hydrophobic molecules will not interact with water and
always have repulsive attitude toward water and let the protein in the folded
form and finally such type of the folding take place in which molecule have
hydrophobic core and all hydrophilic molecules in periphery of the folded
structure(Berezovsky et al. 2001)
Free energy and entropy (G, E)
Gibbs free energy (G) is also defined as the amount of the energy which is
free and this can also be defined as the measure of unstableness and this
measured by simple equation
G= H-TS
G=free energy, H=enthalpy, S=entropy, T= temperature
Entropy (S) is defined as degree of randomness in any system, in terms of
protein folding this can be define as the measure of the possible structure in
the protein molecule at that value of the entropy. Entropy is responsible for
the possible out come of protein as much high would be entropy as much
high number of the confirmation would form. To minimise the entropy in the
protein folding various force come in play like hydrogen bonds, salt bridges,
disulphide bonds etc. these bonds help to reduce the entropy and favour
protein folding(Brady, Sharp 1997)BOLTZMANN worked on the entropy and
give us a very useful relation between atomic theory and entropy. Boltzmann
proposed an equation which shows that entropy in any unfolded protein
structure is equal to the product of his constant(KB) and natural log of number
of all possible states which any protein can adopt(S)
S = KB * NATURAL LOG (S)
KB=Boltzmann constant
Protein folding prefers low value of entropy and follows that smallest path in
which value of entropy is low. At different value of the entropy different
number of structure could be present and finally all structure will vanished
and one structure would be present at the minimum value of entropy, which is
our native structure (Weikl, Dill 2003)
Enthalpy (H) is also responsible for protein folding. This is also known as the
measure of the total energy of the system including internal energy (U). P and
V are the pressure and volume of system
H= U+P*V
So, we should search for such structure which have low value of enthalpy
then the free energy, because which structure have low value of enthalpy that
will have low value of free energy too(Brockwell, Smith & Radford 2000)
Gibbs free energy equation is the single solution for all protein folding
problem. Gibbs equation shows that in case of protein folding, stable state will
have minimum value of “G”. unfolded state have higher free energy then the
folded one and protein folding have many intermediates, This stage is less
populated stage and have maximum energy in the whole system, all above
mention factor in the Gibbs equation adjust and finally provide such folded
state in which value of “G” is minimum and this is known as fully folded and
stable confirmation(Finkelstein, Badretdinov 1997)
(www.biologyonline.org/articles/statistical_thermodynamics_ta
king_walk.html)
Chaperones
Chaperones are bio molecules which participate in the protein folding.
Proteins need assistance in the folding and binds with cofactors .which allow
them to fold properly .these cofactor known as chaperones. These
chaperones bind with the protein as N terminus of the protein formed and
leave ribosome and until and unless protein gain his fully active 3D state and
become functional(Tomala, Korona 2008) Chaperones are not only helping in
the correct protein folding but they also help protein to maintain its correct 3D
structure and prevent them to unfold .these molecules comes in to play when
cell is “under stress” due to favourable conditions are not present and they
also known as the H.S.P (heat shock protein).these molecular chaperones
are HSP40(Dnaj), HSP60(GroEl), HSP70(Dnak) etc.(Rikhvanov, Romanova
& Chernoff 2007)
Copied from Yon, Betton 1991
Mode of action
“Chaperones recognise non native protein structure
by their exposed hydrophobic regions”
Chaperones action is driven by ATP and for the activity of the chaperones
assistance protein folding need of energy, which is provided by the ATP.
These chaperones bind with the intermediate and unfolded protein structure
by the utilisation of the ATP intermediates or random coil structure are
unfolded and again they are fold in the correct 3D structure (NATIVE
structure).
Chaperones molecules are task specific that are different molecules perform
different functions. For example:
HSP70/40
They prevent aggregation and misfolding of newly synthesised protein
molecules.
HSP60
They unfold intermediate and then fold them properly in to native structure
Chaperones are like catalyst, they enhance rate of protein folding and assist
protein folding to native structure and after the formation of the native
structure they separated. As like catalyst they required energy for the initiation
of the process
Chaperones activity is much specialised, in stress condition protein get
mutated, denature, and aggregate. Which may cause some wrong expression
and code for some disease .In such situations they are enough capable to
provide personalised treatment to different protein (Yon, Betton 1991)They
can easily point out that which protein intermediate structure need to be
degraded and which protein intermediate to be stabilise in the native structure
and path should be follow for this process. In some mutation destabilise
protein can be easily stabilized by HSP70 and some over expression of the
specific chaperones. For example: In bacteria HSP70 bind with the protein
polypeptide chain during translation after the synthesis some protein released
for expression and some may attached for some specialised folding and most
destabilise polypeptide chain degraded by chaperones (Tomala, Korona
2008)
Chaperones activity is not simple they have multiple steps in their folding
mechanism. Some chaperones required some other chaperones intermediate
as substrate and then they provide native structure.
Hsp90/70 mechanism chain
Some chaperones may responsible for disease. it has been found that
HSP90 enhance cancer development because many mutated protein mature
in the presence of the HSP90 and cause cancer , repressing HSP90 such
type of cancer can be able to prevent but problem associated with this is due
to this action some non mutagenic protein will be degrade and not express
and cause problem. But on other side HSP70 acts as good repressor in
neurodegenerative disease and prevent this disease in the fruit flies (Tomala,
Korona 2008)
Models of protein modelling and structure
prediction
Plaxco and co-worker model:
This model shows that high degree of correlation between folding rate and
structural properties of protein explain on the basis of contact order (CO).
This can be cross validated from various experiments that folding rate and
contact order are dependent to each other.
,
L is the sequence length
N is the total number of inter-residue atomic contacts
ΔLij is the sequence separation of contacting residues i and j
Kuznetsov and rackovsky showed that structural based determinants can
serve as good determinants of folding rate and many other researchers
searching for which structural and sequence based determinants can serve
as unique predictor of folding rate (Shakhnovich 2006)
Dokholyan and co-workers model:
They use simple protein model and find out transition state of src homology
3(SH3) to find out contribution of each amino acid in transition state. They
calculate Φ value and on this basis they find high correlation between
simulation and experimental Φ value .in the end of their experimental model
they conclude that L24 and G24 are two most important residues in the
folding of proteins
Physics and bioinformatics based models:
Physics models are very helpful to understand protein folding rate and route
to folding. These physics based models help to understand the various forces
and their dynamics in protein folding. These models help to understand:
Conformational changes in protein
Mechanism of folding, enzyme catalysis, mode of action protein
Response to ph salt and denaturants(Brockwell, Smith & Radford 2000)
Bioinformatics is very important tool to find out the structure and folding
pattern of the protein molecules. In this we add our computer based program
along with these physics model and within the fraction of time provide us 3D
structure of protein. Various databases on web are present which contain
information regarding proteins only like NCBI, PUBMED etc. these databases
contain all information about proteins by the comparison of our unknown
sequence using bioinformatics tools with these databases we can find out
possible structure and folding pattern and helpful in drug discovery, possible
remedy against disease etc.
Now, how collectively these factors works
After having the knowledge of these factors now we can easily understand
how they act and result to the fully folded 3D structure. Primary structure of
protein code for 3D structure and all above factors participate to provide a
functional unit. initially primary structure of protein are made of different type
of amino acids on the poly peptide back bone and just after the production of
the N-terminus protein folding starts and secondary structures alpha helix and
beta sheets are formed.
In alpha helix all amino acid chain remain in the periphery of the helix and this
structure formed due to hydrogen bonding and di-sulphide bonding (Trevino,
Scholtz & Pace 2007)
After the formation of secondary structure, tertiary structure these
hydrophobic interaction, hydrogen bonding and charge on the molecule come
it to the play now this protein molecule structure fold in such a way to
minimise all these forces and try to give an stable confirmation to
protein(Chen et al. 2008)
Free energy and entropy act simultaneously. After attachment of these
secondary structures stability is not uniform in the whole tertiary structure.
There may be possibility that two stable structures are joining by unstable and
less stable strands and in that case folding take place in such a way to
minimise the free energy of the system. This type of stage is known as the
intermediate stage. In this stage all amino acids are attached in the structure
but the entropy of the system is high and due to which this show high
presence of free energy and may have the millions of the possibilities of the
intermediate structure. Now protein starts folding from intermediate to the
stable or native structure by minimising the interaction between the
molecules. Due to which all the hydrophobic or non polar amino acid come in
the centre of the structure and the formation of hydrophobic core take place
of 3D structure and all polar or hydrophilic molecules come at the periphery of
the 3D structure(Chen et al. 2008). Out of millions of the possibilities there
are many path which favour this folding in term of having minimum energy
and protein molecule select that path which is shortest and this may contain
several steps in folding and ultimately leads to the formation of native 3D
structure
Copied from Ma, Chen & Zhang 2007
There is always some equilibrium in some folded and intermediates state and
molecules may be aggregate. this depend upon the pH, Temp, Pressure and
denaturation agents and the protein structure destabilise it start affecting
other native protein structure in this stage chaperones act as cofactors and
help in the and maintaining native structure of protein(Ma, Chen & Zhang
2007)
SUMMARY
In the protein folding 3D structure of protein plays an important role in the
protein expression and their function. Information regarding protein folding is
present in the primary structure of the protein which bioenergetics can be
determined by bound amino acid. Hydrophobic forces play an important role.
They all concentrate at the centre of the molecules and hydrophilic at the
periphery of the structure. Hydrogen bonding plays an important role due to
which all polar molecules bound with the surrounding medium in cell and
makes protein molecule structure rigid and compressed.
Protein folding is a spontaneous process in which entropy of the molecule is
decreasing and finally provides a folded structure. Protein native structure
must have low value of Gibbs free energy, entropy, and enthalpy and lower
the value, higher will be the degree of protein stability. Protein may unfold due
to the presence of the unfavourable condition like temp, pressure, pH, and
denaturing agent. In such a case special type of molecules help in the folding
known as chaperones and helpful in maintaining native structure of protein.