Abstract
Incorporating structural elements of thermostable homologs can greatly improve the thermostability of a mesophilic protein. Despite the effectiveness of this method, applying it is often hampered. First, it requires alignment of the target mesophilic protein sequence with those of thermophilic homologs, but not every mesophilic protein has a thermophilic homolog. Second, not all favorable features of a thermophilic protein can be incorporated into the structure of a mesophilic protein. Furthermore, even the most stable native protein is not sufficiently stable for industrial applications. Therefore, creating an industrially applicable protein on the basis of the thermophilic protein could prove advantageous. Amylosucrase (AS) can catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the lone energy source. However, industrial development of AS has been hampered owing to its low thermostability. To facilitate potential industrial applications, the aim of the current study was to improve the thermostability of Deinococcus geothermalis amylosucrase (DgAS) further; this is the most stable AS discovered to date. By integrating ideas from mesophilic AS with well-established protein design protocols, three useful design protocols are proposed, and several promising substitutions were identified using these protocols. The successful application of this hybrid design method indicates that it is possible to stabilize a thermostable protein further by incorporating structural elements of less-stable homologs.
Highlights
Life flourishes almost everywhere on earth, from hydrothermal vents in the deep-sea to the tops of the Himalayas, from rain forests to the hot sands of the Sahara desert, and even from the boiling waters of hot springs to the cold ice field of Antarctica
AcAS has the most proline residues, whereas Deinococcus geothermalis amylosucrase (DgAS), the most stable AS identified so far, has only one more proline than Neisseria polysaccharea amylosucrase (NpAS). This demonstrates that the total proline number alone is not a good indicator of stability, a point stressed in our previous work [17], where we found that the proline number for each individual domain of the stable DgAS is not necessarily more than that of NpAS, the total proline number of NpAS is six lower than that of DgAS
During this study, the sequence and structure of DgAS was compared with those of Deinococcus radiodurans (DrAS), NpAS and the newly identified AcAS, and it was discovered that DgAS has favorable structural properties that can, in part, account for its superior stability
Summary
Life flourishes almost everywhere on earth, from hydrothermal vents in the deep-sea to the tops of the Himalayas, from rain forests to the hot sands of the Sahara desert, and even from the boiling waters of hot springs to the cold ice field of Antarctica. Cellular and cytoplasmic components, proteins, must achieve thermostability [2] For this reason, much effort has been directed towards understanding how proteins from thermophilic organisms retain their structure and function at elevated temperatures [3,4,5,6,7]. Much effort has been directed towards understanding how proteins from thermophilic organisms retain their structure and function at elevated temperatures [3,4,5,6,7] Such understanding is essential for a theoretical description of the physicochemical principles underlying protein folding and stability, but it is critical for designing proteins that can work at high temperatures or are more resistant to unfolding at certain working temperatures. Németh and colleagues improved the Tm of a cellulose C by 3°C [12], and in our opinion this approach is significantly more effective than well-established experimental approaches such as library screening and random site-directed mutagenesis
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