Our understanding of proteins is based on experimental investigations typically performed on purified samples which have been extracted from their native environment. In the case of membrane proteins, which perform major biological functions, these proteins are first extracted from the native environment and then processed so to remove all additional biological material. Our challenge is to perform a single molecule investigation on membrane proteins embedded in their natural environment of neurons from central nervous system, such as cortical and/or hippocampal neurons.
We have performed protein pulling experiments, i.e. single molecule force spectroscopy (SMFS) of membrane proteins embedded in their native/physiological environment, at room temperature and in the presence of the physiological medium. We have also developed a new tool for the analysis of the Force-distance (F-d) curves obtained in SMFS experiments providing an almost automatic classification of the obtained SMFS data in a set of different clusters. Our method provides a classification of the possible unfolding pathways, present during the unfolding of membrane proteins. A comparison with the available proteomic investigations of the membrane proteins of hippocampal and cortical neurons identifies good molecular candidates of the obtained clusters of F-d curves. Our work shows that it is possible to perform single molecule investigations of membrane proteins avoiding purification keeping these proteins in their natural environment. This single molecule analysis suggests a significant structural heterogeneity not seen in usual X-ray crystallography and/or Cryo-EM investigations.