Computational study of retro Trp-cage

Vymětal J.¹, Bathula S.R.², Žídek L.² , Černý J.³, Sklenář V .² and Vondrášek J.¹

¹Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16610 Prague 6, Czech Republic

²Laboratory of Biomolecular Structure and Dynamics, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic

³Institute of Biotechnology, Academy of Sciences of the Czech Republic, v. v. i., Vídeňská 1083, 142 20 Praha 4, Czech Republic

Prediction of the three-dimensional structure of a protein from sequences unrelated to any previously known structure belongs to the most complicated tasks in computational structure biology. The generally accepted fact that sequence determines protein fold can be further elaborated in search for its ground or more general rules for sequence - structure relationship. We examined the case of reverse sequence of a known protein which keeps the sequential distance between all amino acids as in the original protein but their order is reversed. We hypothesized that such arrangement may be still potent in preserving of native inter-residual contacts between amino acids and thus retain the same fold as the original sequence.

We used reversed Trp-cage miniprotein sequence (retro Trp-cage) in testing of proposed hypothesis. The three-dimensional structures of the retro Trp-cage sequence were obtained by the current most successful in silico predictions methods - Robetta and PEP-FOLD. Folding simulations using molecular dynamics were also used for structural characterization of the reverse Trp-cage sequence and its folding and dynamical stability. NMR study of the synthetic miniprotein provided that there is no stable 3D structure in pure water, however it could be induced by addition of trifluorethanol (TFE). The experimental NMR structure of the retro Trp-cage in TFE resembled the general Trp-cage fold but showed distinct packing of the protein core and different inter-residual contacts which were not identical with the original Trp-cage protein neither with any predicted model. The presence of TFE promoted formation of helix involving the same amino acids as in the original Trp-cage molecule.

Our results revealed that reversed sequence of Trp-cage miniprotein is not able to maintain all native Trp-cage contacts and folds differently than the original structure. Although the helical preferences in the reverse sequence was preserved (confirmed by both theoretical as well as experimental studies) it can be only manifested in suitable environment. All modeling methods and simulations predicted the occurrence of helix but overestimated its stability and did not provide the correct model of the miniprotein. Obviously, structural prediction of proteins and peptides on the edge of stability or in non-water environments remains challenging themes in molecular modeling.