Andrea Scirè

The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.

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 [Lys(18)-->Gly (K18G) and Arg(82)-->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 p(2)H-dependent. All proteins revealed the existence of a molten globule-like state. At p(2)H 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 p(2)H, 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 p(2)Hs tested for E. coli Trx. The thermal stability of the proteins was in the following order at all p(2)Hs tested: BacTrx>R82E>K18G> E. coli Trx, and was lower for each protein at p(2)H 8.4 than at neutral or acidic p(2)Hs. The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p(2)Hs tested, whereas they were present in R82E and E. coli Trx samples only at p(2)H 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.

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability

Glutamine‐binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L‐glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X‐ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of α‐helices than β‐sheets in the protein both in the absence and in the presence of glutamine. Generalized two‐dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications. Proteins 2005.

STRUCTURE–ACTIVITY RELATIONSHIP ON FUNGAL LACCASE FROM RIGIDOPORUS LIGNOSUS: A FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.

Structural and thermal stability characterization of Escherichia coli D-galactose/D-glucose-binding protein.

The effect of temperature and glucose binding on the structure of the galactose/glucose‐binding protein from Escherichia coli was investigated by circular dichroism, Fourier transform infrared spectroscopy, and steady‐state and time‐resolved fluorescence. The data showed that the glucose binding induces a moderate change of the secondary structure content of the protein and increases the protein thermal stability. The infrared spectroscopy data showed that some protein stretches, involved in α‐helices and β strand conformations, are particularly sensitive to temperature. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein is well represented by a three‐exponential model and that in the presence of glucose the protein adopts a structure less accessible to the solvent. The new insights on the structural properties of the galactose/glucose‐binding protein can contribute to a better understanding of the protein functions and represent fundamental information for the development of biotechnological applications of the protein.

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis : The binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D‐trehalose/D‐maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20°–100°C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90°C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90°C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of α‐helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport‐related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications. Proteins 2006.

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric β‐glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ssβ‐gly), Thermosphaera aggregans (Taβ‐gly), and Pyrococcus furiosus (Pfβ‐gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ssβ‐gly and Taβ‐gly (temperatures of melting 98.1 and 98.4°C, respectively) were less stable than Pfβ‐gly, which maintained its secondary structure even at 99.5°C. The thermal denaturation of Pfβ‐gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D‐structures of these enzymes supported the experimental results: Ssβ‐gly and Taβ‐gly are stabilized by a combination of ion‐pairs networks and intrasubunit S‐S bridges while the increased stability of Pfβ‐gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea. Proteins 2007.

Two-dimensional IR correlation spectroscopy of mutants of the β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus identifies the mechanism of quaternary structure stabilization and unravels the sequence of thermal unfolding events

Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion pairs compared with mesophilic glycoside hydrolases. The ion pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. In the present study, the structure and thermal stability of the wild-type beta-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were examined by FTIR (Fourier-transform IR) spectroscopy. The FTIR data revealed small differences in the secondary structure of the proteins and showed a lower thermostability of the mutant proteins with respect to the wild-type. Generalized 2D-IR (two-dimensional IR correlation spectroscopy) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild-type, indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilizes the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. This evidence gives support to the current theories on the mechanism of ion-pair stabilization in proteins from hyperthermophilic organisms.

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier‐transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as α‐helices and β‐sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to α‐helices whose content resulted in 5% of the total secondary structure of the protein. The short α‐helix found in N. coriiceps metallothionein showed an onset of denaturation at 30°C and a Tm at 48°C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the α‐helix and in the metal‐thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time‐resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins. Proteins 2002;46:259–267.

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima.2.Molecular organization and structural stability

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscopic observations with specific secondary structural elements. The data determined the presence of a buried beta-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding of bacterial binding protein family members and their potential biotechnological applications.