Cooperation between a Salt Bridge and the Hydrophobic Core Triggers Fold Stabilization in a Trp-Cage Miniprotein

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• 作者：Pé ; ter Hud&aacute ; ky ; P&aacute ; l Str&aacute ; ner ; Viktor Farkas ; Gyö ; rgyi V&aacute ; radi ; G&aacute ; bor Tó ; th ; Andr&aacute ; s Perczel
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：January 22, 2008
• 年：2008
• 卷：47
• 期：3
• 页码：1007 - 1016
• 全文大小：470K
• 年卷期：v.47,no.3(January 22, 2008)
• ISSN：1520-4995

文摘

Miniproteins are adequate models to study various protein-structure modifying effects suchas temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residueTrp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W. et al. (2002)Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable atrelatively low temperatures. Here we report a systematic investigation of structural factors influencingthe stability of Tc5b by solving its solution structure in different environments, varying temperature, andpH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions,selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigatedby NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. Theseobservations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b,in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability andadopts a stable fold at physiological temperature.