具有36個胺基酸的雞絨毛蛋白(HP36)為一個小的螺旋蛋白，由三個α-螺旋結構所組成，而其三個α-螺旋則會形成一疏水核心(hydrophobic core)以穩定整體結構。該疏水核心由三個苯丙胺酸(phenylalanine) Phe47，Phe51，Phe58和一個纈胺酸(valine)Val50所組成。由於HP36的特殊結構及其快速的折疊(folding)速度，使得它成為一個研究蛋白質折疊的優良模型。在以往的研究當中顯示，將氟化後的苯丙胺酸置換於疏水核心後可以提升其疏水性進而增加蛋白質的穩定性。氟原子自身也可以改變芳香環上的電子密度分布，進而誘發、增強或削弱各種靜電作用力。然而，過多的置換亦可能造成額外的立體障礙效應進而降低蛋白質結構的穩定性。
在本實驗當中，我們分別使用了在四號位置用氟原子取代的苯丙胺酸(4-fluorophenylalanine)及甲基取代的苯丙胺酸(4-methylphenylalanine)來置換Phe47, Phe51及Phe58；所合成的變異蛋白質有:F51Z、F51Z/F58Z、F47Z/F51Z/F58Z、F58X、F51Z/F58X、和 F51X/F58Z。其中Z代表4-氟苯丙胺酸，而X代表4-甲基苯丙胺酸。我們先利用far-UV CD光譜與NMR光譜來鑑定其結構，並確定所有衍生物皆與原生態(WT)具有相似之構形。接著再以變溫實驗與化學誘導變性實驗來探究其穩定性，並得出其穩定性之排序: F51Z > F51X/F58Z ~ F51Z/F58Z ~ F51Z/F58X > WT > F47Z/F51Z/F58Z ~ F58X。為了更加了解其穩定性之趨勢，我們利用了Discovery Studio這套軟體來進行簡易的模擬運算，所使用的力場為CHARMm，並得出各胜肽的能量最適化後的結構。由各個結構當中可清楚看到疏水核心中的苯丙胺酸的相對位置，而由該相對位置我們計算了之間的作用力，並得出各種作用力如polar...π和凡得瓦作用力對其穩定性有一定的影響力。此外，我們也認為在不同位置的置換可能會誘發不適當的立體障礙效應，因而降低蛋白質的穩定性。
由我們的研究當中可以發現該核心的作用力遠比我們所想像的還要複雜，彼此互相牽涉了許多作用力，沒有單一的作用力能夠決定最後的穩定性。雖然就現階段而言我們仍無法全數闡明它們之間的詳細交互關係，不過我們在此研究當中提出了一種更加細節的觀點來探討HP36的折疊作用力，希望這樣的結果能對未來的蛋白質設計能有更多的了解與幫助。

HP36, the helical subdomain of villin headpiece, known as one of the smallest naturally occurring cooperatively folded proteins. Due to its small size, three-helical topology and rapid folding rate, HP36 is a good model for computational and theoretical studies of protein folding. This subdomain contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, Phe58), and one valine residue (Val50). Polar…π interactions and hydrophobic interactions have been shown to be critical factors to stabilize HP36, and also been proposed to be general forces in stabilizing small proteins. By mutating Phe47, Phe51 and Phe58 with fluorinated or methylated phenylalanine, we can modulate the aromatic-aromatic interactions between these phenylalanine residues and the folding stability of HP36.
To investigate the consequences of incorporated fluorinated or methylated phenylalanine into the hydrophobic core of HP36, here we synthesized wild type HP36 (WT) and its six variants F51Z, F51Z/F58Z, F47Z/F51Z/F58Z, F58X, F51Z/F58X, and F51X/F58Z, where the Z represents 4-fluorophenylalanine, and the X represents 4-methyl- phenylalanine. Far-UV CD and NMR spectra indicate that all of the mutants fold into a similar conformation to that of WT. The stability has also been measured by thermal and chemical denaturation experiments for each variant. The stability order was found as: F51Z > F51X/F58Z ~ F51Z/F58Z ~ F51Z/F58X > WT > F47Z/F51Z/F58Z ~ F58X. In order to elucidate this interesting tendency, we further performed simple simulations by the Discovery Studio software. By applying CHARMm force field and adopted basis Newton-Raphson method to get their energy optimized final structures, we were able to examine the orientation between these three Phe residues, and to calculate the interaction energies.
Both the outcomes of the simulations and the final structures are quite fascinating: the orientations of the three Phe residues show edge-face or semi-face-face geometries to each other, which might induce different electrostatic interactions including polar…π interaction and/or other π...π stacking interactions. The introduced fluorine atom and methyl group not only increased the hydrophobicity but also induced the rearrangement of electron density of the phenyl rings, which would enhance or decrease the electrostatic interactions. Besides, we also believe that steric effects play another important factor to modulate the stabilities.
Our study showed us that the interactions among the hydrophobic core are much more complicated than we imagined, and no single factor would overwhelm others. Although we are not able to clarify all of them at the present stage, we did provide a more detailed point of view to appreciate the interactions among the HP36 hydrophobic core. Our results should be useful and helpful for the future protein design study.

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