Optimal Salt Bridge for Trp-Cage Stabilization

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• 作者：D. Victoria Williams ; Aimee Byrne ; James Stewart ; Niels H. Andersen
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：February 22, 2011
• 年：2011
• 卷：50
• 期：7
• 页码：1143-1152
• 全文大小：961K
• 年卷期：v.50,no.7(February 22, 2011)
• ISSN：1520-4995

文摘

Gai and co-workers [Bunagan, M. R., et al. (2006) J. Phys. Chem. B 110, 3759鈭?763] reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 [Hudaky, P., et al. (2008) Biochemistry 47, 1007鈭?016]; the authors suggested that [D9E]-TC5b presented a more compact and melting resistant structure because of the 鈥渙ptimal distance between the two sides of the molecule鈥? Nonetheless, the authors reported essentially the same circular dichroism (CD) melting temperature, 38 卤 0.3 掳C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by nuclear magnetic resonance and CD with the following mutations: [D9E], [D9R,R16E], [R16O], [D9E,R16O], [R16K], and [D9E,R16K]. Of these, the [D9E] mutant displayed the smallest acidification-induced change in the apparent T_m. In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9鈭扲16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence of salt bridge formation is also provided for a swapped pair, the [D9R,R16E] mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence that was cited as supporting increased fold stability for [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded states rather than a more favorable salt bridge. Our study also provides evidence of other Trp-cage stabilizing roles of the R16 side chain.