Effects of a Novel Disulfide Bond and Engineered Electrostatic Interactions on the Thermostability of Azurin
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- 作者：Anna Tigerströ ; m ; Frederick Schwarz ; Gö ; ran Karlsson ; Mats Ö ; kvist ; Carmen Á ; lvarez-Rú ; a ; Dennis Maeder ; Frank T. Robb ; and Lennart Sjö ; lin
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 5, 2004
- 年：2004
- 卷：43
- 期：39
- 页码：12563 - 12574
- 全文大小：448K
- 年卷期：v.43,no.39(October 5, 2004)
- ISSN：1520-4995
文摘
Identification and evaluation of factors important for thermostability in proteins is a growingresearch field with many industrial applications. This study investigates the effects of introducing a noveldisulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurinfrom Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and noveltemperature-dependent atomic displacement factors from crystal data collected at elevated temperatures.The atomic displacement parameters describe the molecular movement at higher temperatures. Thethermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry.Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting aflexible loop with small 
-helix (D62C/K74C copper-containing mutant), increased the Tm by 3.7 
Ccompared with the holo protein. Furthermore, three mutants were designed to introduce electrostaticinteractions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between twoseparate 
beta2.gif" BORDER=0 ALIGN="middle">-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore,D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability.The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to1.5 and 1.8 Å resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has alsobeen completed to 1.8 Å resolution. These structures support the selected design and provide valuableinformation for evaluating effects of the modifications on the thermostability of holo azurin.
-helix (D62C/K74C copper-containing mutant), increased the Tm by 3.7 Ccompared with the holo protein. Furthermore, three mutants were designed to introduce electrostaticinteractions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between twoseparate beta2.gif" BORDER=0 ALIGN="middle">-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore,D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability.The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to1.5 and 1.8 Å resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has alsobeen completed to 1.8 Å resolution. These structures support the selected design and provide valuableinformation for evaluating effects of the modifications on the thermostability of holo azurin.