Modulating the Interactions in a Small Helical Protein HP36 by Nonnatural Amino Acids
| Autor: | Hsu, Wei-Lin, 許維麟 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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