The Folding Pathway of Onconase Is Directed by a Conserved Intermediate

Abstract

A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

“…This made it possible to detect the first 6 amide hydrogens to be involved in native-like structure after 180 seconds. Almost all of the amide hydrogens of residues stabilized after 1200 seconds are the same as those first detected in the previous work (17). Therefore, the present experiments have enabled us to study much earlier events in the folding of onconase.…”

“…Because folding was so rapid in this study (17), it was not possible to obtain much detail about early folding events. By contrast, in our oxidative-folding study, by allowing S-S/SH interchange to occur along the 3S U -to-3S F transition, the formation of native structure occurs on a much longer time-scale, 1200 seconds.…”

“…In an earlier study of the disulfide-bond-intact folding pathway of guanidine-denatured ONC, quench flow methods coupled with H/D exchange and 2D-NMR spectroscopy identified 31 amide hydrogens of stabilized residues from the protein backbone as the earliest detectable structure in ONC after 250 ms and folding was complete in ~5 seconds (17). Because folding was so rapid in this study (17), it was not possible to obtain much detail about early folding events.…”

Stability and folding of amphibian ribonuclease A superfamily members in comparison with mammalian homologues

“…Furthermore, the RNase A homologues differ in the number of proline residues they contain (up to seven), and in particular, in the number of cis ‐proline residues (i.e. cis Xaa–Pro peptide bonds), which significantly affects the folding of the respective proteins .…”

“…(Right) Unfolding and refolding kinetics of ONC were determined manual mixing ( k U , slow , ●, k f, medium , ), by stopped‐flow ( k f, fast ,▽, k f, medium , ○, and k f, slow , □), or double‐jump experiments ( k U , fast , ▼), respectively (data from Ref. , reproduced with permission).…”