Abstract
Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so.
Highlights
Thioredoxins (TRXs) are small molecular weight proteins (,14 kDa), which are present in all organisms from bacteria to mammals
The acquisition of compactness (SEC), tertiary and secondary structures (CD), and the burial of hydrophobic residues (ANS) occur simultaneously in the three PsTRXs; that is, the three TRXs from Pisum sativum behave in their pH denaturation, showing the presence of a molten-globule-like protein. Whether these species are monomers; the fact that they elute at larger volumes than the corresponding folded TRX, suggests protein-column interactions, probably due to solvent-exposure of hydrophobic residues, and the possibility of aggregation to bury those solventexposed hydrophobic patches
The presence of this partially unfolded form in the three PsTRXs do not agree with the acidic denaturation observed in TRX of E. coli, where the secondary and tertiary structure of the protein does not change from pH 3.0 to 7.0 [62]; notwithstanding these facts, kinetic intermediates during the folding of E. coli TRX have been described by using several techniques [62,63,64]
Summary
Thioredoxins (TRXs) are small molecular weight proteins (,14 kDa), which are present in all organisms from bacteria to mammals. As a consequence TRXs are associated with a number of human pathologies, such as cancer, cardiac disease or viral infections; recently, TRXs have been used as potential therapeutic regulators of cell growth, apoptosis and inflammation [5]. Since they were first described by Reichard and co-workers [6], the number of TRX members identified in prokaryotes and eukaryotes has increased dramatically. The TRX types m, x and y are related to prokaryotic TRXs, while types f, h and o are related to eukaryotic organisms [18,19,20]
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