Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

Abstract

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.

Unfolded protein exhibits antiassociation activity toward the 50S subunit facilitating 70S ribosome dissociation

“…Several studies have demonstrated that the ribosome is itself capable of assisting in the folding of proteins spanning a wide range of folds and functions [1]. The protein-folding ability of the ribosome appears to be a universal one and has been demonstrated with ribosomes isolated from a wide range of sources including eubacteria, archaebacteria, eukaryotes (yeast, rat liver, and wheat germ), rabbit reticulocyte, bovine mitochondria, and mitochondria of the parasite Leishmania donovani [2][3][4][5][6][7][8][9]. These studies have also established that the chaperoning ability of the bacterial ribosome originates from the peptidyl transferase center that resides in domain V of the 23S rRNA of the bacterial large ribosomal subunit.…”

The Chaperone-Active State of HdeB at pH 4 Arises from Its Conformational Rearrangement and Enhanced Stability Instead of Surface Hydrophobicity

“…Urea, a chemical denaturant, primarily disrupts hydrophobic interactions and hydrogen bonds, while guanidinium chloride can additionally interfere with electrostatic interactions. 31,34 pH, a relatively milder denaturant, mainly affects the ionization states of certain amino acids in a protein. Therefore, pH-sensitivity of a protein inform us about the contribution of ionic interactions in its stability.…”

“…Therefore, pH-sensitivity of a protein inform us about the contribution of ionic interactions in its stability. 31 On the other hand, thermal denaturation disrupts noncovalent bonds and destabilizes the folded state by increasing the protein's kinetic energy and mainly disrupting hydrophobic interactions. 31 Understanding these differences helps researchers interpret the observed changes in protein structure and function upon denaturation.…”

“…31 On the other hand, thermal denaturation disrupts noncovalent bonds and destabilizes the folded state by increasing the protein's kinetic energy and mainly disrupting hydrophobic interactions. 31 Understanding these differences helps researchers interpret the observed changes in protein structure and function upon denaturation. Therefore, we attempted to understand the conformational states and the stability of acid-stress chaperones under varying stress conditions.…”

Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength

“…40 This folding activity has been shown to promote productive folding from denatured and MG states. [41][42][43] Concerning apoflavodoxin, first results show that at physiological ionic strength the ribosome restrains formation of the off-pathway MG of F44Y apoprotein and forces the nascent chain towards the native state. 44 As MG off and the on-pathway folding intermediate of F44Y apoflavodoxin are concurrently present at physiological ionic strengths (Fig.…”