Hydrophobic And Hydrophilic Interaction In Protein Folding Biology Essay
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Review the mechanism of protein folding
Module: Enzyme Technology and Biocatalysts
Module leader: Prof. Steve Forsythe
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.
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)
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"
GLUTAMIC ACID "E"
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=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)
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
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)
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:
They prevent aggregation and misfolding of newly synthesised protein molecules.
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
Î”Lij 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 Î¦ value and on this basis they find high correlation between simulation and experimental Î¦ 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)
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.
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