Volume Exclusion Effect as a Driving Force for Reverse Proteolysis: IMPLICATIONS FOR POLYPEPTIDE ASSEMBLAGE IN A MACROMOLECULAR CROWDED MILIEU

Balajee R Somalinga,Rajendra P Roy

doi:10.1074/jbc.m207974200

Balajee R Somalinga, Rajendra P Roy

Open Access

https://doi.org/10.1074/jbc.m207974200

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Abstract

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.

Highlights

IntroductionThe study of biochemical processes in macromolecular crowded media has attracted considerable attention in recent years because of the awareness that reactions in living cells occur in a highly crowded milieu of background macromolecule (proteins, carbohydrates, nucleic acids, lipids, etc.) solutes, and these inert macromolecules may influence the kinetics and equilibrium of reactions through excluded volume effect arising from nonspecific steric repulsion between molecules [1,2,3,4,5]
The study of biochemical processes in macromolecular crowded media has attracted considerable attention in recent years because of the awareness that reactions in living cells occur in a highly crowded milieu of background macromolecule solutes, and these inert macromolecules may influence the kinetics and equilibrium of reactions through excluded volume effect arising from nonspecific steric repulsion between molecules [1,2,3,4,5]
Proteases must catalyze synthesis of the peptide bond, as demanded by the principle of microscopic reversibility of reactions, reverse proteolysis is facilitated under thermodynamically favorable situations that do the following: (a) diminish the enthalpic barrier by enhancing protonation of the carboxyl group, (b) reduce the entropy cost of bringing the two reacting peptides in stereo-chemical proximity, and (c) render stability to the newly synthesized peptide bond

Summary

Introduction

The study of biochemical processes in macromolecular crowded media has attracted considerable attention in recent years because of the awareness that reactions in living cells occur in a highly crowded milieu of background macromolecule (proteins, carbohydrates, nucleic acids, lipids, etc.) solutes, and these inert macromolecules may influence the kinetics and equilibrium of reactions through excluded volume effect arising from nonspecific steric repulsion between molecules [1,2,3,4,5]. The hydrolytic or degradative reactions are likely to be somewhat hindered in the presence of macromolecular crowding relative to the dilute solutions It follows that molecular crowding or confinement under favorable conditions may promote shifting of the equilibrium of such reactions toward synthesis. The protease-catalyzed ligation reactions involving native polypeptide segments (totally unprotected fragments) in vitro are promoted in the presence of organic cosolvents and generally for those participating fragments whose reacting termini are brought into proximity through noncovalent associations (10 –12) This principle of shifting the peptide bond equilibrium is based on the notion that whereas the enthalpic barrier (ionization of the carboxyl) to peptide bond synthesis is lowered in the presence of organic cosolvent, the entropic barrier is overcome by the proximity of the reacting ends. Reverse Proteolysis in a Crowded Milieu situations proteases might catalyze the synthesis of a peptide bond in vivo

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