Abstract
The X‐ray structure of [N‐acetyl]‐apamin has been solved at 0.95 Å resolution. It consists of an 1‐7 N‐terminal loop stabilized by an Asn‐β‐turn motif (2–5 residues) and a helical structure spanning the 9–18 residues tightly linked together by two disulfide bonds. However, neither this accurate X‐ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn^2^ and Glu^7^ amide signals upon Glu^7^ carboxylic group ionization. Thus, apamin and its [N‐acetyl], [Glu^7^Gln], [Glu^7^Asp], and [Asn^2^Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu^7^ carboxylate group is responsible for the large downfield shifts of the Asn^2^ and Glu^7^ amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn^2^ and Glu^7^ amide protons as well as the N‐terminal α‐amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn^2^Abu] analogue, revealed an essential role of Asn^2^ in the β‐turn stability and the cis/trans isomerization of the Ala^5^‐Pro^6^ amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu^7^ carboxyl group. However, neither destabilization of the β‐turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn^2^ and Glu^7^ residues appeared essential for the N‐terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 447–462, 2007.
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