1H and 15N assignment and secondary structure of the double K18G/R82E mutant of
Alicyclobacillus acidocaldarius thermostable thioredoxin

Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability.

Glu85 in the Escherichia coli thioredoxin, which is localized in the loop
between beta4 and beta5, was substituted with the Arg present in the
corresponding position in Bacillus acidocaldarius thioredoxin. This suggested
that it could play an important role in the structure and thermostability of
this protein owing to its involvement in numerous interactions. The effects of
the mutation on the biophysical properties were analysed by circular dichroism,
spectrofluorimetry and limited proteolysis, supported by molecular dynamics
data. As modelling predicted, an increase in stability for E85R due to
additional H-bonds between the beta5 and alpha4 regions was observed.

Pedone E, Saviano M, Rossi M, Bartolucci S.
A single point mutation (Glu85Arg) increases the stability of the thioredoxin from
Escherichia coli.
Protein Eng. 2001 Apr;14(4):255-60.

A single point mutation (Glu85Arg) increases the stability of the thioredoxin from Escherichia coli.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium
growing optimally at 333 K, is the first Trx described to date from a moderate
thermophilic source. To understand the molecular basis of its thermostability,
the three-dimensional structure in the oxidized form was determined by NMR
methods. A total of 2276 1H-NMR derived distance constraints along with 23
hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a
protocol employing simulated annealing followed by restrained molecular
dynamics and restrained energy minimization. BacTrx consists of a well-defined
core region of five strands of beta-sheet, surrounded by four exposed
alpha-helices, features shared by other members of the thioredoxin family. The
BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx)
determined by X-ray crystallographic diffraction, and a number of structural
differences were observed that may contribute to its thermostabilty. The
results of structural analysis indicated that protein stability is due to
cumulative effects, the main factor being an increased number of ionic
interactions cross-linking different secondary structural elements and clamping
the C-terminal alpha-helix to the core of the protein.

Nicastro G, De Chiara C, Pedone E, Tato M, Rossi M, Bartolucci S.
NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible
determinants of protein stability.
Eur J Biochem. 2000 Jan;267(2):403-13.

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to
homogeneity by anion-exchange chromatography and gel-filtration chromatography,
based on its ability to catalyse the dithiothreitol-dependent reduction of
bovine insulin disulphides. The protein has a molecular mass of 11577 Da,
determined by electrospray mass spectrometry, a pI of 4.2, and its primary
structure was obtained by automated Edman degradation after cleavage with
trypsin and cyanogen bromide. The sequences of known bacterial Trxs were
aligned at the active site: BacTrx has an identity ranging from 45 to 53% with
all sequences except that of the unusual Anabaena strain 7120 Trx (37%
identity). The gene coding for BacTrx was isolated by a strategy based on PCR
gene amplification and cloned in a plasmid downstream of a lac-derived promoter
sequence; the recombinant clone was used as the expression vector for
Escherichia coli. The expression was optimized by varying both the time of cell
growth and the time of exposure to the inducer isopropyl
beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total
cytosolic protein. CD spectra and differential scanning calorimetry
measurements demonstrated that BacTrx is endowed with a higher conformational
heat stability than the Trx from E. coli. Nanogravimetry experiments showed a
lower content of bound water in BacTrx than in E. coli Trx, and a transition
temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model
of the oxidized form of BacTrx was constructed by a comparative molecular
modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as
reference proteins. Increased networks of ion-pairs and shorter loops emerged
as major features of the BacTrx structure compared with those of the template
proteins. The findings are discussed in the light of the current knowledge
about molecular determinants of protein stability.

Bartolucci S, Guagliardi A, Pedone E, De Pascale D, Cannio R,
Camardella L, Rossi M, Nicastro G, de Chiara C, Facci P, Mascetti G,
Nicolini C.
Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in
Escherichia coli and molecular modelling.
Biochem J. 1997 Nov 15;328 ( Pt 1):277-85.

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

Alicyclobacillus acidocaldarius thioredoxin

The Crystal Structure Of The K18g Mutant Of The Thioredoxin From Alicyclobacillus Acidocaldarius

The Crystal Structure Of The Mutant R82e Of Thioredoxin From Alicyclobacillus Acidocaldarius

Solution Structure Of The Thioredoxin From Bacillus Acidocaldarius

Solution Structure Of The K18gR82E ALICYCLOBACILLUS Acidocaldarius Thioredoxin Mutant

thioredoxin [Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446]

thioredoxin [Alicyclobacillus acidocaldarius subsp. acidocaldarius Tc-4-1]

thioredoxin [Alicyclobacillus acidocaldarius]

RecName: Full=Thioredoxin; Short=Trx [Alicyclobacillus acidocaldarius subsp. acidocaldarius]

Alicyclobacillus acidocaldarius (Bacillus acidocaldarius)
Alicyclobacillus acidocaldarius subsp. acidocaldarius

TORSION ANGLE DINAMICS AND RESTRAINED ENERGY MINIMIZATION