“Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST‐NMR Experiments, and Molecular Dynamics Calculations

Abstract

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

“…Low sample volume (0.1–1 mL) and concentration (µM range) requirements make this method a sensible choice for the rapid determination of protein conformation in order to verify if an expressed, purified protein is correctly folded, or how different mutations may affect its structure and thermal stability [32]. The ECD spectrum of PAF[Pc] at 25 °C was highly similar to that reported previously for this protein [26] and other disulphide bridged, β-structured proteins [33] (Fig. 4a).…”

“…The lack of NMR signals from unlabelled PAF[Pc] in single or double labelled samples proved that the incorporation of stable isotopes was close to 100%. The comparison of the 15 N- 1 H and 13 C- 1 H heteronuclear single quantum coherence (HSQC) spectra of the 15 N/ 13 C-PAF[Pc] sample to the previously recorded NMR data of 15 N-labelled or 15 N/ 13 C-labelled PAF generated in P. chrysogenum wild-type Q176 demonstrated the absolute spectral identity of these protein samples [13, 26] (Fig. 6).…”

“…The preferential codon usage of P. chrysogenum was taken into account for gene modification. The amino acids Phe25, Ile26, Phe31 and Tyr48 form a hydrophobic patch on the surface of PAF [13, 26]. This motif is assumed to play a major role in the interaction of this AP with the plasma membrane of target fungi [13, 26].…”

“…The amino acids Phe25, Ile26, Phe31 and Tyr48 form a hydrophobic patch on the surface of PAF [13, 26]. This motif is assumed to play a major role in the interaction of this AP with the plasma membrane of target fungi [13, 26]. To address this assumption, we exchanged Phe31 and Tyr48 with the uncharged, polar residues asparagine and glutamine, respectively and created PAF F31N and PAF Y48Q (Fig.…”

“…1). In PAF, these cysteines form three disulphide bonds in abcabc pattern, which is essential for proper folding and full antifungal activity [13, 25, 26]. A similar folding is highly probable for NFAP [4].…”