The pH-dependent tertiary structure of a designed helix-loop-helix dimer

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Abstract

Background: De novo designed helix-loop-helix motifs can fold into welldefined tertiary structures if residues or groups of residues are incorporated at the helix-helix boundary to form helix-recognition sites that restrict the conformational degrees of freedom of the helical segments. Understanding the relationship between structure and function of conformational constraints therefore forms the basis for the engineering of non-natural proteins. This paper describes the design of an interhelical HisH+-Asp- hydrogen-bonded ion pair and the conformational stability of the folded helix-loop-helix motif. Results: GTD-C, a polypeptide with 43 amino acid residues, has been designed to fold into a hairpin helix-loop-helix motif that can dimerise to form a four-helix bundle. The folded motif is in slow conformational exchange on the NMR timescale and has a well-dispersed 1H NMR spectrum, a narrow temperature interval for thermal denaturation and a near-UV CD spectrum with some fine structure. The conformational stability is pH dependent with an optimum that corresponds to the pH for maximum formation of a hydrogenbonded ion pair between HisH17+ in helix I and Asp27- in helix II. Conclusions: The formation of an interhelical salt bridge is strongly suggested by the pH dependence of a number of spectroscopic probes to generate a well-defined tertiary structure in a designed helix-loop-helix motif. The thermodynamic stability of the folded motif is not increased by the formation of the salt bridge, but neighbouring conformations are destabilised. The use of this novel design principle in combination with hydrophobic interactions that provide sufficient binding energy in the folded structure should be of general use in de novo design of native-like proteins. © Current Biology Ltd.

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Dolphin, G. T., & Baltzer, L. (1997). The pH-dependent tertiary structure of a designed helix-loop-helix dimer. Folding and Design, 2(5), 319–330. https://doi.org/10.1016/S1359-0278(97)00043-6

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