Issue 20, 2018

Protein stabilization by tuning the steric restraint at the reverse turn


Reverse turns are solvent-exposed motifs in proteins that are crucial in nucleating β-sheets and drive the protein folding. The solvent-exposed nature makes reverse turns more amenable to chemical modifications than α-helices or β-sheets towards modulating the stability of re-engineered proteins. Here, we utilize van der Waals repulsive forces in tuning the steric restraint at the reverse turn. The steric restraint induced upon N-methylation of the i+1–i+2 amide bond at the reverse turn results in well-folded and stable β-sheets in aqueous solution at room temperature. The developed superactive turn inducing motif is tolerant to a wide variety of functional groups present on coded amino acids making the designed turn fully compatible with bioactive loops in proteins. We demonstrate that the steric restraint and the functional groups at the reverse turn act in synergy to modulate the folding of re-engineered β-sheets. Introduction of the turn motifs onto a three-stranded β-sheet protein, Pin 1 WW domain, resulted in various analogs showing a cooperative two-state transition with thermal stability (TM) ranging from 62 °C to 82 °C. Despite modulating the stability of Pin 1 variants by ∼2.8 kcal mol−1 (ΔΔGf), the native fold in all the protein variants was found to be unperturbed. This structural stability is brought about by conformational preorganization at the engineered reverse turn that results in strong intramolecular hydrogen bonds along the three dimensional structure of the protein. Thus, this simple loop engineering strategy via two amino acid substitution provides us a “toolkit” to modulate the stability of β-sheet containing peptides and proteins in aqueous solution that will greatly expand the scope of de novo protein and foldamer design.

Graphical abstract: Protein stabilization by tuning the steric restraint at the reverse turn

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Article information

Article type
Edge Article
05 Dec 2017
24 Apr 2018
First published
24 Apr 2018
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2018,9, 4600-4609

Protein stabilization by tuning the steric restraint at the reverse turn

P. Lahiri, H. Verma, A. Ravikumar and J. Chatterjee, Chem. Sci., 2018, 9, 4600 DOI: 10.1039/C7SC05163H

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