Depending on how many residues you "shuffle," the structural change could be anywhere from very minor (few changes, especially if the changed residues' side chains are surface exposed) to completely unfoldable. Even changing a few residues could have a major structural impact if those residues are key to the folding process. The fact that the amino acid composition stays the same as the wild-type protein does not improve the likelihood that the structure will be unchanged when you shuffle the sequence. Finally, even if the overall fold of the mutant is essentially the same as the wild-type, they will still be different structurally by definition, even if only one surface side chain is different. Functionally, even such a small difference could have a large impact on aggregation. Consider the case of sickle cell hemoglobin versus wild-type hemoglobin.

Dear Swarup Chakrabarty,

with a conservation of 92 residues out of approx 200 it is likely that naturally occuring proteins will share fold and possible function as well..

However if you generate random sequences substituting the non-conserved regions, I think your protein may fail to fold correctly, let alone function correctly.

Let me explain in more detail my thoughts:

You need to define more precisely what you mean by conserved residues.Are they conserved by identity, or by similarity? Are you attempting to maintain disulphide bridges -these are very important for maintaing structure - and in your case it is likely that you will have 2-4 disulphide bridges stabilizing your structure.

For the non-conserved regions, you should divide it into solvent exposed and buried regions. For the solvent exposed region you should adapt a substitution strategy that conform with the expected Amino acid distribution found on the surface of soluble proteins. Conversely, for the buried regions that are non-conserved you should a substitution strategy that conform with the buried Amino acid distribution. But also make sure that Amino acids in alfahelices are substituted with Amino acids that has a high alfahelix propensity and similarly for Amino acids in beta strands. Finally the substitution you select should maintain the residue volume.

If you substitue 'brute force' without the above considerations you are bound to generate sequences where a number of buried residues will be charged (eg Lys, Arg, Glu and Asp). This is known to destabilize folded structures dramatically. Only ion pairs are found buried. Surface substitutions are rather flexible with respect to charge - whereas function may change in response to a charge change. For buried residues, there is a well known propensity for secondary structure preference.This may assist you in selecting a likely substituion from one that is expected to conflict with known folding preferences. Please also observe that the residue volume should be maintained. If you substitute a buried Gly with a Trp , you are generating steric conflicts, which may prevent the protein from folding.

*Please find a selection of papers from my own Work that may help you in your further studies:

 A happy new year to you

Steffen

Article A critical view on conservative mutations

Article Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

Article Scale-Free Behaviour of Amino Acid Pair Interactions in Fold...

Data Protein engineering the surface of enzymes

I agree with Dr. Shapiro and Dr. Petersen.

Good luck!

I also agree with the other statements.

Even if single mutations are likely to be tolerated especially at the surface of your protein, the combination of several varied side-chains is likely to destroy the fold capacity. These random changes will prevent the formation of efficient side-chain interactions which are necessary as an thermodynamic driving force for folding.

I agree in general with Adam.

Mainly, keeping the composition the same doesn't save anything (if I understood correctly, you mean, that you had e.g. 15 Ala and 5 Val and you will keep those, just put them in other position).

On the other hand, few changes that do not conserve the composition, but are "close enough" may be tolerable (e.g. changing Val to Leu, Ile or Ala). But of course it depends on case to case scenario, changing Ala to Ile somewhere inside may be detrimental, because it will not simply fit, but many changes on the surface may be OK.

If you're looking for increasing stability, look for cavities in your protein, hydrophobic amino acids on the surface, same charge close to each other (like Arg and Lys in proximity). There are few other empirical rules (like polarity of the alpha-helices).

Thanks alot for your suggestions :)