Reviewing The Mechanism Of Protein Folding Biology Essay

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Scope of protein folding. Proteins are the biomolecules which plays and pivotal role in this living world. They are responsible for expression of certain characters in different types of cells and around 50% of the total cell dried mass. Proteins are the chain of amino acids which bind with polypeptide backbone and then fold in a unique 3D (native structure) by which expression of the protein take place, Various forces and factors are responsible for the protein folding and due to the minute difference in the protein composition structure will change and right expression of the protein will not take place and cause disease. Many of the disease like Alzheimer's; Parkinson, cystic fibrosis etc (Brignall et al, 2007) are in human body due to not proper folding of proteins. For proper expression of protein these amino acid should be in their unique 3D structure. Some time protein required assistance in folding, molecules which help in the folding known as chaperones. These molecules help in the folding of the certain protein molecules and also prevent the unfolding of the molecules and keep them in accurate structure.

In this assignment we will study about the mechanism of the protein folding, affect of various factors which influence the protein folding and developments in thermodynamics, kinetics of protein folding in past two decade.


Protein folding is very complex mechanism and great development has been done in last 20years, by the use of various techniques like N.M.R, X RAY CRYSTELLOGRAPHY, MASS SPECTROMETERY, SPECTROPHOTOMETER etc. we manage to find the structure of the protein and also find how actually protein folding takes place. In this protein folding various factors act together and finally construct specific 3D structure to protein. Protein folding is very fast process and it may take milliseconds to seconds.

Due to high speed of folding this is not possible to find out the each and every possible conformation in fraction of time. LEVINTHAL stated that each protein have millions of pathway by which a desired 3D pattern achieved and express. By study of intermediates we can under stand the mechanism or pattern on which protein folding works. But this is impossible to find out all possible structure of protein molecules.

Protein folding study used denaturants which help to denature the protein and then left him in unfolded stage. They use high PH, TEMP, PRESSURE etc which stop folding at intermediate stage and help in understanding folding pattern by study of these intermediates. There are many possible structures in between the unfolded and fully folded structure. Fully folded structure also known as NATIVE structure

Protein folding inside cell is known as in-vivo and out side cell means in lab conditions is known as in-vitro. 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 and also help protein molecule to retain its folded confirmation and make them stable.

In-vitro concentration of macromolecules is very less around 1% of originally present in cell. So inside cell these molecules also affect protein stability and make them more stable than in vitro ( Jessica A.O rumfeldt et al, 2008).

Protein folding may be in two stage or multistage. This depend upon which type of sequence is folding and a single point mutation can change folding from single to multistage and vice-versa .Some previous studies shows that protein sequence made up of less than 80 amino acid prefer two stage folding and large protein sequence having amino acid above then 130 prefer multi stage folding. Later on they prove that sequence rich in F and G amino acid prefer two stage folding and sequence rich in C, H, L and R amino acid prefer multistage folding as per the table given below (bin-guang ma et al, 2007).


Hydrophobic and hydrophilic interaction

Proteins are made of the 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(saul r. Trevino et al,2006).























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 its suffer 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 (Robert l. Baldwin,2007).


All amino acids have different energetic in secondary structure. There are many chameleon sequence 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

2007., Protein Folding: Then and Now

Yiwen Chen,* Feng Ding,* Huifen Nie,* Adrian W. Serohijos,* Shantanu Sharma,* Kyle C. Wilcox,* Shuangye Yin,* and Nikolay V. Dokholyan*†

Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599

† To whom correspondence should be addressed: Nikolay V. Dokholyan, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina 27599. Fax: 919-966-2852. Email:[email protected]



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 (y.s djikave et al , 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(igor n. berezovsky,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



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(g patric brady,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)


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 (Thomas r. weiki et al, 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(david j brockwell et al,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 (alexi v finkelstein, 2006) .


Chaperones are bio molecules which participate in the protein folding. Protein 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 (katarzyna tomala,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.(Eugene g. rikhvanov et al, 2007)

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 have their specific work all type of chaperones molecules not doing the same work they have their specialised task .

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 (jeannine m yon et al,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 mutational destabilise protein can easily denature by the normal concentration of HSP70 and mutated protein can easily stabilise by some over expression of the specific chaperones 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 (katarzyna tomala,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 act as good repressor in neurodegenerative disease and prevent this disease in the fruit flies ( katarzyna tomala,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 validate 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

2009, Protein Folding Thermodynamics and Dynamics: Where Physics, Chemistry and Biology Meet

Eugene Shakhnovich

Eugene Shakhnovich, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge MA 02138;

[email protected]

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 model help to understand how various force and 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

The Protein Folding Problem

Ken A. Dill,1,2 S. Banu Ozkan,3 M. Scott Shell,4 and Thomas R. Weikl5

1 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

2 Graduate Group in Biophysics, University of California, San Francisco, California 94143; email:[email protected]

3 Department of Physics, Arizona State University, Tempe, Arizona 85287; email:[email protected]

4 Department of Chemical Engineering, University of California, Santa Barbara, California 93106; email:[email protected]

Bioinformatics is very impotent 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

Start 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 (saul r. Trevino et al,2006).

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 (yiwen 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 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(cui yan tan et al,2005). 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

There is always some equilibrium in some folded and intermediates state and molecules may be aggregate. this depend upon the PH, TEMP, PRESSURE AND DENATURING 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 (bin-guang ma et al,2007)


Conclusion drawn from the above discussed information is that:-

In the protein folding 3D structure of protein plays an impotent 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 and all amino acid which were hydrophobic. 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 non 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 as much low value of these parameter would be that much high degree of the stability of the structure.

Protein may unfold due to the presence of the unfavourable condition like TEMP, PRESSURE , PH, DENATURING AGENT , in this case special type of molecules help in the folding known as CHAPERONES and helpful in maintaining native structure of protein .

Hydrophobic force result in the folding of the molecules, entropy act in the unfolding and the native structure is the resultant of the both of them