Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in thermal denaturation pathway of the proteins: an infrared spectroscopic study

The structure of thioredoxin from Alicyclobacillus acidocaldarius (previously named Bacillus acidocaldarius) (BacTrx) and from Escherichia coli (E. coli Trx) was studied by Fourier-transform IR spectroscopy. Two mutants of BacTrx [Lys18→Gly (K18G) and Arg82→Glu (R82E)] were also analysed. The data revealed similar secondary structures in all proteins, but BacTrx and its mutants showed a more compact structure than E. coli Trx. In BacTrx and its mutants, the compactness was p2H-dependent. All proteins revealed the existence of a molten globule-like state. At p2H 5.8, the temperature at which this state was detected was higher in BacTrx and decreased in the different proteins in the following order: BacTrx>R82E>K18G>E. coli Trx. At neutral or basic p2H, the molten globule-like state was detected at the same temperature in both BacTrx and R82E, whereas it was found at the same temperature in all p2Hs tested for E. coli Trx. The thermal stability of the proteins was in the following order at all p2Hs tested: BacTrx>R82E>K18G>E. coli Trx, and was lower for each protein at p2H 8.4 than at neutral or acidic p2Hs. The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p2Hs tested, whereas they were present in R82E and E. coli Trx samples only at p2H 5.8. The results indicated that a single mutation might affect the structural properties of a protein, including its propensity to aggregate at high temperatures. The data also indicated a possible application of Fourier-transform IR spectroscopy for assessing molten globule-like states in small proteins.