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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Ensemble Fit of Conformational Equilibria of RestrainedPeptides to NMR Data. Dependence on Force fields:Amber8 vs ECEPP/3J. Ciarkowski 1 , S. Łuczak 1 , M. Oleszczuk 2 , and J. Wójcik 31 Faculty of Chemistry, University of Gdańsk, 80-952 Gdańsk, Poland; 2 Department ofBiochemistry, University of Alberta, T6G 2H7CA, Canada; 3 Institute ofBiochemistry and Biophisics, PAS, 02-106, Warsaw, PolandIntroductionSearching an optimal method for conformational analysis of flexible peptides, we used twodermorphin analogs Tyr-D-Daa-Phe-Daa-NH 2 comprising combinations of D-Dab 2 (α,γdiaminobutyryl)with Lys 4 , 1, and of D-Dap 2 (α,β-diaminopropionyl) with Orn 4 , 2. Theyhad side chain amino groups bridged with >C=O, restraining 1 and 2 with 16- and 14-membered rings and leading to potent and impotent μ/δ opioid peptides, respectively [1].The conformational equilibria of 1 and 2 were found by fitting their ensembles to the NOEand J NMR data using the method introduced by Groth, et al. [2], implementing themaximum entropy principle to avoid overfit. The latter turns up when a physical quantity(here equilibrium), is fitted to data charged with inherent statistical uncertainty (here NOEand J). In the procedure quoted, the overfit is dispersed by the introduction of the scaledentropy term (scaling factor α), favoring uniform distribution of conformers [2]. Typically,for α=0 (no entropy term), a fit delivers a few conformations, the standard deviation (SD)of the objective function is ≤ than that transposed from the measured SD in J and NOE, andwe arrive at effectively fitting a good share of noise (overfit). On the opposite side, e.g.α≈2, thousands of conformers are found, SD grows larger and more realistic, χ 2 testbecomes excellent. For flexible peptides, an objective is a compromise between these twooptions to be reached by trial-and-error, featured with SD ≥ than that transposed fromparameter errors and with a handy conformational distribution meeting χ 2 criterion.Results and DiscussionThe Authors [1] provided us with their NMR NOE- and J-data in H 2 O/D 2 O, and with theresults of fitting conformational equilibria of 1 and 2 to their NMR data using an ensemblegenerated by means of EDMC/ECEPP/3 (rigid-valence geometry) methodology [2]. Wesubmitted 1 and 2 to the 4 ns molecular dynamics (AMBER v.8, flexible-valence) in theperiodic boundary conditions at constant pressure, using the locally enhanced sampling(LES-MD, 5 copies) for more efficient sampling. The time step was 2 fs, the coordinatessaved every 2000 steps and put together. In the time range 0.48-4 ns every fifth set wascollected, resulting with 880 conformers per 1 and/or 2.Earlier use of EDMC/ECEPP methodology [2] by some of us resulted with 7structures for 1 and 11 for 2 [1], hereby applied as a reference. The structures were receivedfrom the fits restoring the NMR parameters to ~94% and not employing the entropy term,α=0 [1], see Table 1. Currently, using fits to the LES-MD/AMBER [3] ensemble, weselected the entropy factor α=0.001 by trial-and-error. The choice resulted in 154 structuresFig. 1. Macrocycle-fitted overlap of LES-MD/AMBER (grey) for EDMC/ECEPP (black)structures: left – for 1 and;right – for 2.598