Maximum entropy approach to the determination of solution conformation of flexible polypeptides by global conformational analysis and NMR spectroscopy - Application to DNS1-c-[D-A(2)bu(2), Trp(4),Leu(5)]-enkephalin and DNS1-c-[D-A(2)bu(2), Trp(4), d-Leu(5)]enkephalin

A method is proposed to determine the conformational equilibrium of flexibl e polypeptides in solution, using the data provided by NMR spectroscopy and theoretical conformational calculations. The algorithm consists of the fol lowing three steps: (i) search of the conformational space in order to find conformations with reasonably low energy; (ii) simulation of the NOE spect rum and vicinal coupling constants for each of the low energy conformations ; and (iii) determining the statistical weights of the conformations, by me ans of the maximum-entropy method, in order to obtain the best fit of the a veraged NOE intensities and coupling constants to the experimental quantiti es. The method has been applied to two cyclic enkephalin analogs: DNS1-c-[d -A(2)bu(2),Trp(4),Leu(5)]enkephalin (ENKL) and DNS1-c-[d-A(2)bu(2),Trp(4),d -Leu(5)]enkephalin (ENKD). NMR measurements were carried out in deuterated dimethyl sulfoxide. Two techniques were used in conformational search: the electrostatically driven Monte Carlo method (EDMC), which results in extens ive search of the conformational space, but gives only energy minima, and t he molecular dynamics method (MD), which results in a more accurate, but al so more confined search. In the case of EDMC calculations, conformational e nergy was evaluated using the ECEPP/3 force field augmented with the SRFOPT solvation-shell model, while in the case of MD the AMBER force field was u sed with explicit solvent molecules. Both searches and subsequent fitting o f conformational weights to NMR data resulted in similar conformations of t he cyclic part of the peptides studied. For both ENKL and ENKD a common fea ture of the low-energy solution conformations is the presence of a type II' or type IV beta-turn at residues 3 and 4; the ECEPP/3 force field also giv es a remarkable content of type III beta-turn. These beta-turns are tighter in the case of ENKL, which is reflected in different distributions of the d-A(2)bu((NH)-H-gamma)... d-A(2)bu(CO) and d-A(2)bu((NH)-H-gamma)... Gly(3) (CO) hydrogen-bonding distances, indicating that the d-A(2)bu((NH)-H-gamma) amide proton is more shielded from the solvent than in the case of ENKD. T his finding conforms with the results of temperature coefficient data of th e d-A(2)bu((NH)-H-gamma) proton. It has also been found that direct (MD) or Boltzmann (EDMC) averages of the observables do not exactly conform with t he measured values, even when explicit solvent molecules are included. This suggests that improving force-field parameters might be necessary in order to obtain reliable conformational ensembles in computer simulations, witho ut the aid of experimental data.