Question

Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allow certain key protein structural features to be formed. Can you identify an amino acid(s) that would be affected by this reducing environment? How would it be affected, and what structural features would be sensitive to this environment? Can you discuss the implications of this from a standpoint of recombinant protein expression? (500 words)

Answer 1

Cysteine is the amino acid which gets affected by reducing environment .

Cysteine has a thiol side chain which forms disulfide bond with another cysteine in a protein. When the protein gets into reducing environment the disulfide bonds will get reduced and the disulfide bonds will break. This will result in the formation of two thiol groups unbound to each other.

– S–S–       –SH + HS–     

The tertiary and quaternary structures of proteins are reinforced with disulfide bonds if present. When the disulfide bonds get reduced it may lead to disruption of the three dimensional structure of proteins.

During the folding process the conformational entropy of proteins get reduced . disulfide bonds if present contribute majorly to this decrease in entropy . on exposing the protein to reducing environment the breaking of disulfide bonds cause partial increase in the entropy and may lead to unfolding/ partial denaturation.

Implications from the standpoint of recombinant protein expression:

Suppose we have cloned a thermo-stable protein in bacteria. This protein will be rich in cysteine-cysteine disulfide bonds. When the bacteria expresses the foreign recombinant protein in its system, the formation of a biologically-active native structure of the protein will require that its primary structure gets folded correctly into the tertiary/quaternary structure. This will require the formation of disulfide bonds during the folding process. However, if the protein is transported to the periplasmic space of the bacteria, its disulfide bonds will get reduced as a result of which the protein will lose its active form, either completely or partially.