Synergetic Effects of Alanine and Glycine in Blob-Based Methods for Predicting Protein Folding Times

The polypeptide PGlyAlaGlu was prepared with 20 mol % glycine (Gly), 36 mol % d,l-alanine (Ala), and 44 mol % d,l-glutamic acid (Glu) and labeled with the dye 1-pyrenemethylamine to yield a series of Py-PGlyAlaGlu samples. The fluorescence decays of the Py-PGlyAlaGlu samples were analyzed according to the fluorescence blob model (FBM) to obtain the number N_blob^exp of amino acids (aa's) encompassed inside the subvolume V_blob of the polypeptide probed by an excited pyrene. An N_blob^exp value of 29 (±2) was retrieved for Py-PGlyAlaGlu, which was much larger than for any of the copolypeptide PGlyGlu or PAlaGlu prepared with either Gly and Glu or Ala and Glu, respectively. The continuous increase in N_blob^exp with decreasing side chain size (SCS) from 10 aa's for PGlu to 16 aa's for PAlaGlu and 22 aa's for PGlyGlu was used earlier to define the reach of an aa and determine the groups of aa's that could interact with each other along a polypeptide backbone according to their SCS. These groups of aa's, referred to as blobs, led to the implementation of blob-based models (BBM) to predict the folding time τ_F^theo,BBM of 145 proteins, which was found to match their experimental folding time τ_F^exp with a relatively high 0.71 correlation coefficient. Nevertheless, the much higher N_blob^exp value found for Py-PGlyAlaGlu compared to all other pyrene-labeled polypeptides studied to date indicates that the reach of aa's along a polypeptide sequence is affected not only by SCS but also by synergetic effects between different aa's. Following this new insight, a revised BBM was implemented to predict τ_F^theo,BBM for 195 proteins assuming the existence or absence of synergies to control the interactions between aa's along a polypeptide sequence. Similarly good correlation coefficients of 0.71 and 0.74 were obtained for a direct 1:1 comparison of τ_F^exp and τ_F^theo,BBM for the 195 proteins without and with synergies, respectively. This result suggests that synergetic effects between different aa's have little effect on τ_F^theo,BBM predicted from BBM underlying the robustness of this methodology.