What we have learned, at the molecular level, about the mechanisms that allow proteins (enzymes) to be stable and operate at their high physiological temperature is the combined effect of reduction of the size of external loops and maximising the stabilising interactions (hydrogen bonds, salt bridges in particular) plus having a large hydrophobic core.

This allows the enzyme to attain the flexibility required for function (catalysis) at the high temperature the organism thrives at (see for example Irimia et al [2004], J. Mol. Biol. 335, 343-356, "The 2.9 A Resolution Crystal Structure of Malate Dehydrogenase from Archaeoglobus fulgidus: Mechanisms of Oligomerisation and Thermal Stabilisation" on the most thermostable malate dehydrogenase know to date).

By consequence, in order to render a protein mesophilic starting from a thermophile you'd need to go the opposite way: lengthen the outside loops, reduce the number of stabilising interactions and reduce the size of the hydrophobic core.

This is likely to be a trial and error process, that is propose a few mutations (it is preferable to have the 3D structure at hand to do this in a "rational" manner), produce the modified protein, check its thermal stability properties, if feasible determine its 3D structure to see what in fact happened in the modified protein i.e. learn from your mistakes, improve on your first proposal by proposing additional mutations etc.

And do keep in mind that nature has had millions of years to allow for this adaptation, whereas you have at most a lifetime to devise such a protein adaptation in a "clever" way. Unless you want your children to work on the same topic as you are :) . But the good news (for you), as was mentioned previously, is that most mutations have deleterious effects: they destabilise proteins. But you want to do this in a clever, programmed fashion.

Can you explain why you want to convert the thermostable protein into thermolabile protein. One possible way is to modify the disulfide bonds in the protein to make it thermolabile protein if it is not affecting the function of the protein.

Most of the mutations are destabilizing  -  if you make mutation

in the protein sequence away from the active site likely outcome will be

the less stable protein.

Dear Kedar,

Generally if you make mutations in the core of the protein, those mutations would lead to reduced stability. The idea is that the core of a protein has specific structure and  by making mutations in the core you perturb the core of the protein. The amount of perturbation would depend upon the amount of the change as you are going from one amino acid to another amino acid. This reduced stability might give you a mesophilic protein.

Another strategy could be disrupting the electrostatic interactions. If some salt bridges are known for your protein which impart stability to the protein, then you can try disrupting those salt bridges by mutating the charged residue(s) with neutral residue(s).

Hope you will get the mesophilic protein by either of the above strategies.

Best,

Jogender

What we have learned, at the molecular level, about the mechanisms that allow proteins (enzymes) to be stable and operate at their high physiological temperature is the combined effect of reduction of the size of external loops and maximising the stabilising interactions (hydrogen bonds, salt bridges in particular) plus having a large hydrophobic core.

This allows the enzyme to attain the flexibility required for function (catalysis) at the high temperature the organism thrives at (see for example Irimia et al [2004], J. Mol. Biol. 335, 343-356, "The 2.9 A Resolution Crystal Structure of Malate Dehydrogenase from Archaeoglobus fulgidus: Mechanisms of Oligomerisation and Thermal Stabilisation" on the most thermostable malate dehydrogenase know to date).

By consequence, in order to render a protein mesophilic starting from a thermophile you'd need to go the opposite way: lengthen the outside loops, reduce the number of stabilising interactions and reduce the size of the hydrophobic core.

This is likely to be a trial and error process, that is propose a few mutations (it is preferable to have the 3D structure at hand to do this in a "rational" manner), produce the modified protein, check its thermal stability properties, if feasible determine its 3D structure to see what in fact happened in the modified protein i.e. learn from your mistakes, improve on your first proposal by proposing additional mutations etc.

And do keep in mind that nature has had millions of years to allow for this adaptation, whereas you have at most a lifetime to devise such a protein adaptation in a "clever" way. Unless you want your children to work on the same topic as you are :) . But the good news (for you), as was mentioned previously, is that most mutations have deleterious effects: they destabilise proteins. But you want to do this in a clever, programmed fashion.

The reverse i.e. making a mesophilic enzyme to a thermophilic one is tougher. An interesting way is to make chimeras using a mesophilic and thermophilic proteins. The challenge is not just the thermostability of the resultant protein, other parameters kcat, Km, oligomerization, stability... all get affected when you do any mutagenesis. One should always check all these parameters, it is rare that only one aspect of a protein gets modified when a mutation is made (most of the time we look for what we want and move on). See this paper for an interesting outcome (Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization. .Proteins. 2008 Dec;73(4):1010-20)

Another option is to target via iterative saturation mutagenesis (ISM) those residues displaying the highest B-factor values (usually obtained from X-ray data). As summarized here:

http://link.springer.com/protocol/10.1007%2F978-1-4939-1053-3_7

"The B-factors give a measure of the relative vibrational motion of atoms and thus, electron density. Because it is known that thermostable enzymes are usually rigid, targeting flexible residues displaying the highest B-factors can be a useful strategy to enhance the thermal stability or organic solvent tolerance of enzymes. The number of flexible residues can be very large depending on the enzyme, but those displaying the highest B-factors are generally preferred."

The B-FIT iterative test severs to improve the thermostability of a (usually mesophilic) enzyme, so one can think on doing exactly the inverse on a thermostable enzyme to get a mesophilic one via ISM. For an example of the experimental protocol, see here:

http://www.nature.com/nprot/journal/v2/n4/full/nprot.2007.72.html

Yes, it is possible. To be efficient, you will need to develop a high throughput screen in order to select the desirable clones. What type of protein are you working with and does it have an enzymatic function that may be exploited to develop a screen/selection?

I guess you have found this publication in the meantime from reading the suggestions carlos made?

DOI: 10.1002/bit.22202

In this case, the group  of M. Reetz (B-FIT program) has engineered thermolability.

Usefull references within.