Setting the basis for the study of intermediate conformers of human neuroserpin by Double Electron Electron Resonance

The Double Electron Electron Resonance (DEER) is an innovative technique that allows to measure the distance distribution between two spin labels within a range of 2-8 nm. This technique does not require crystalline samples, thus it is possible to determine the position of two different spin labelled domains of intrinsically flexible macromolecular systems. Our idea is to determinate the structural details of the intermediate conformers of human neuroserpin (hNS) by DEER. hNS is a protein that in the native state is folded in a metastable conformation. In particular conditions, hNS can either adopt a more stable conformation with the reactive centre loop (RCL) inserted into a β-sheet (latent conformer) or insert the RCL into the β-sheet of a neighbour hNS molecule thus forming dimers or polymers (polymeric conformers). This research project requires the production of spin labelled hNS with both a single and a double cysteine mutation via Site-Directed Spin Labelling (SDSL). We have already performed a first preliminary experiment on native hNS spin labelled at positions 157 and 296 (see Figure 1). The results are in agreement with the available crystallographic structure of native hNS and show the applicability of the DEER technique to the study of hNS conformers that have not been yet crystallized.