Restrained Molecular Dynamics Procedure for Protein Tertiary Structure Determination from NMR Data: A Lac Repressor Headpiece Structure Based on Information on J‐coupling and from Presence and Absence of NOE's

In recent years a procedure has been developed by which the three-dimensional (3D) structure of biomolecules can be derived from 2D NMR data. This procedure combines model building with restrained energy minimization (EM) and molecular dynamics (MD) techniques. Distance information from NOE's is incorporated in the form of an upper limit distance restraining term that is added to the interatomic potential function. Here, two improvements of the refinement procedure are introduced. First, the information that is contained in empty parts of 2D-NOE spectra is transformed into, so-called, non-NOE's which are modelled by adding a lower limit distance restraining term to the potential function for each non-NOE proton pair. Secondly, the information that is contained in the occurrence of large J-coupling constants for specific dihedrals is modelled by adding a sinusoidal dihedral angle restraining term to the potential function for each dihedral angle with a large J-value. The improved refinement procedure is tested by application of the lac repressor headpiece. Both the inclusion of non-NOE data and the inclusion of J-coupling information markedly improve the result of the restrained MD refinement of headpiece. In the refinement procedure MD simulation is used for searching configuration space. Energy barriers which are too high to be crossed by MD are surmounted by manually changing the model structures on a picture system. The resulting close non-bonded contacts are relaxed by EM. The final structure of headpiece satisfies essentially all 169 NOE and 9529 non-NOE distance constraints as well as the 6Cα – Cβ dihedral angle values corresponding to the measured J-coupling values.