The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C-terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8-20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5-10-fold loss of activity was found for PrRP8-20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTANMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full-length peptide, which exists in an equilibrium between α- and 3₁₀-helix. We demonstrate that PrRP8-20's reduced propensity to form an α-helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α-helical C-terminal region of PrRP is critical for receptor activation. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 273–281, 2013.

Predicting HIV-1 protease/inhibitor binding affinity as the difference between the free energy of the inhibitor bound and unbound state remains difficult as the unbound state exists as an ensemble of conformations with various degrees of flap opening. We improve computational prediction of protease/inhibitor affinity by invoking the hypothesis that the free energy of the unbound state while difficult to predict is less sensitive to mutation. Thereby the HIV-1 protease/inhibitor binding affinity can be approximated with the free energy of the bound state alone. Bound state free energy can be predicted from comparative models of HIV-1 protease mutant/inhibitor complexes. Absolute binding energies are predicted with R = 0.71 and SE = 5.91 kJ/mol. Changes in binding free energy upon mutation can be predicted with R = 0.85 and SE = 4.49 kJ/mol. Resistance mutations that lower inhibitor binding affinity can thereby be recognized early in HIV-1 protease inhibitor development.

EM-Fold was used to build models for nine proteins in the maps of GroEL (7.7 Å resolution) and ribosome (6.4 Å resolution) in the ab initio modeling category of the 2010 cryo-electron microscopy modeling challenge. EM-Fold assembles predicted secondary structure elements (SSEs) into regions of the density map that were identified to correspond to either α-helices or β-strands. The assembly uses a Monte Carlo algorithm where loop closure, density-SSE length agreement, and strength of connecting density between SSEs are evaluated. Top-scoring models are refined by translating, rotating, and bending SSEs to yield better agreement with the density map. EM-Fold produces models that contain backbone atoms within SSEs only. The RMSD values of the models with respect to native range from 2.4 to 3.5 Å for six of the nine proteins. EM-Fold failed to predict the correct topology in three cases. Subsequently, Rosetta was used to build loops and side chains for the very best scoring models after EM-Fold refinement. The refinement within Rosetta's force field is driven by a density agreement score that calculates a cross-correlation between a density map simulated from the model and the experimental density map. All-atom RMSDs as low as 3.4 Å are achieved in favorable cases. Values above 10.0 Å are observed for two proteins with low overall content of secondary structure and hence particularly complex loop modeling problems. RMSDs over residues in secondary structure elements range from 2.5 to 4.8 Å. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 669–677, 2012.