Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement.

单链莫内林 (SCM) 是一种连接莫内林两条链的工程蛋白，莫内林是一种天然甜味蛋白质。这种蛋白质是用作食品和饮料中的糖替代品的有吸引力的候选者，并且具有许多其他应用。因此，产生稳定性提高的 SCM 突变体是拓宽其潜在应用范围的一个活跃的研究领域。在本研究中，我们重点关注 SCM 的 Cys41 残基，它是存在于结构上重要位置的单个半胱氨酸残基。该残基通常被 Ser 取代。然而，这种取代可能会破坏 SCM 的稳定性，因为 Cys41 埋藏在蛋白质的疏水核心中。因此，我们设计了用 Ala、Val 和 Leu 替代该残基的突变体，即 C41A、C41V 和 C41L。我们对这三种突变体、SCM C41S 和野生型 (WT) 进行了表征。差示扫描荧光分析表明，用Ala或Val取代Cys41提高了SCM的热稳定性，而用Ser或Leu取代则降低了其稳定性。对SCM C41A和C41V突变体的晶体结构测定表明，两个突变体的整体结构和第41位残基周围的主链结构与WT几乎相同。另一方面，第 41 个残基附近的氨基酸侧链的方向在 SCM 变体之间有所不同。综上所述，我们的结果表明，用 Ala 或 Val 替代 Cys41 提高了 SCM 的稳定性，并提供了对这种改进的结构基础的深入了解。