Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant:  A Molecular Analysis of Its Reduced Thermal Stability^,

Abstract

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.

Structural and biochemical insights of CypA and AIF interaction

“…Almost complete H N and N resonance assignments of 15 N-HSQC-detected residues of CypA were achieved, as previously reported 34 , based on 3D 15 N-edited NOESY 35 and 15 N-edited TOCSY spectra 36 , acquired with a mixing time of 80 and 50 ms, respectively, in combination with previous assignments reported in literature 37 . The indole H N group of the single Trp residue was unambiguously assigned.…”

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

“…Some excipients stabilize proteins by direct interaction, whereas others by indirect mechanisms such as preferential hydration 6,16. It has been shown that the flexibility of the amide backbone and side‐chain interactions can play a significant role in maintaining protein thermal stability 18–20. In addition, solution conditions such as pH and temperature can have profound effects on immunoglobulin dynamics and stability through alteration of solvent fluctuations 21–22.…”

Mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles

“…It has also been suggested that the stability of thermostable proteins may result from a balance between packing and solubility [54]. Enhanced stability of proteins from hyperthermophilic organisms has been discussed in terms of increased rigidity at room temperature [55-58], but this is not universally supported [59,60]. However, there is a consensus view that the enhancement of thermostability in proteins from thermophiles is due to a complex balance of interactions at numerous sites [15,55,61], and that it is difficult to identify a single common determinant [14,62].…”