Folding and Stabilization of Native-Sequence-Reversed Proteins

Abstract

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

Figure 1

Predicted structures and backbone RMSDs for Protein G’s (a) native β-hairpin (GEWTYDDATKTFTVTE), (b) sequence-reversed β-hairpin (ETVTFTKTADDYTWEG), and (c) modified sequence-reversed β-hairpin (ETVTFTKTDASDDYTWEG). The residues in bold/red font were inserted into the reverse sequence to improve hydrophobic packing within the hairpin’s core. Key residues are rendered as vdW spheres and the residues inserted into the sequence in (c) are indicated with white sticks. Hydrophobic residues in the structure predicted for the reverse sequence are not well packed, leading to subsequent deviations in backbone RMSD over the course of MD simulations (b). By contrast, the (a) native and (c) modified hairpin structures are both stable in solution, experiencing only small fluctuations during simulation.

Figure 2. Secondary structural components of the modified, sequence-reversed Trpzip2 as a function of time.

The hairpin fold remains stable and well-defined for the entire length of the simulation.

Figure 3

(a) Structure of native α3D; hydrophobic residues are shown as vdW spheres. (b) Structure of sequence-reversed α3D. (c) Solvent accessible surface areas (SASAs) of sequence-reversed and native α3D structures and their component hydrophobic residues. Hydrophobic residues within the reverse sequence stay buried throughout the simulation, featuring a SASA comparable to that associated with native α3D (particularly with respect to the crystal structure).

Figure 4

(a) Native structure of Trp-cage (NLYIQWLKDGGPSSGRPPPS). (b) Backbone RMSD of one of the modified, sequence-reversed Trp-cages (SPPPRGSSPGGDKWLQIYLN). The insets provide representative snapshots showing the proline tail deforming the upper helix while trying to bury Trp6. Plotting schemes mirror those used in Fig. 1.

Figure 5

Structures and contact maps for (a) native Protein G and (b) sequence-reversed Protein G. Hydrophobic residues are shown in a stick representation, and residues key to each structure’s hydrophobic core are highlighted in red. The mean minimum residue distances presented in the contact maps are calculated by averaging over the whole 100 ns MD trajectory.