Volume exclusion effect as a driving force for reverse proteolysis. Implications for polypeptide assemblage in a macromolecular crowded milieu.
Macromolecular crowding, in principle, should affect any reaction that is accompanied by significant reduction in excluded volume. Here we have examined the influence of crowding on reverse proteolysis. We show that proteosynthesis of a polypeptide product with an interacting folding motif such as coiled coil is facilitated in crowded media as a consequence of the volume exclusion effect. Further, we demonstrate that crowding could also effect the conversion of a noncovalent protein complex (fragment complementing protein) obtained by limited proteolysis to the native covalent form, but only if the formation of the native protein results in large compaction leading to a substantial volume exclusion effect. Subtilisin-catalyzed reformation of native triosephosphate isomerase (TIM) from multiple fragments is facilitated by crowding. However, a single nick in ribonuclease S (RNase S) could not be ligated under similar conditions. The failure of generation of RNase A from RNase S is consistent with the fact that the crystal structure of the two forms are almost superimposable, and hence no significant difference of volume exclusion exists between reactant (RNase S) and product (RNase A). In contrast, considerable compaction, and consequently large reduction in excluded volume, is attained through the assembly of a TIM barrel structure. Taken together, these results have implications for both in vitro as well as in vivo polypeptide assemblage by reverse proteolysis.
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Balajee R
BR
Somalinga
Rajendra P
RP
Roy
D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D010455:Peptides; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S; D012697:Serine Endopeptidases; D013381:Subtilisins; C028344:glutamyl endopeptidase; D014305:Triose-Phosphate Isomerase