Simulated annealing with restrained molecular dynamics using CONGEN: Energy refinement of the NMR solution structures of epidermal and type‐α transforming growth factors

Abstract

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168;Bassolino-Klimas D et al., 1996, Protein Sci5:593-603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type-a transforming growth factor (hTGFa). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR-derived constraints for mEGF and 424 NMR-derived constraints for hTGFa were used in these energy-refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low-energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., "relaxed") sets of the EGF and hTGFa internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy-refined structures of mEGF and hTGFa are compared with structures determined previously and with structures of homologous proteins determined by NMR and X-ray crystallography.

Simulated annealing with restrained molecular dynamics using a flexible restraint potential: Theory and evaluation with simulated NMR constraints

“…The current implementation of CONGEN also allows use of dihedral angle constraints and J constraints. Successful applications of the general SA protocol described here and the CONGEN computer program to structure determinations of peptides and proteins using real NMR data will be described in detail elsewhere (Newkirk et al, 1994;Jendeberg et al, 1996;Tejero et al, 1996). In addition, the ability to model ensembles of structures with the correct averaging of NOES and vicinal coupling constants has been added to CONGEN recently.…”

“…Accordingly, all of the structures described in these benchmark tests are initiated from extended conformations with randomly assigned initial velocities. In related work using real NMR distance constraints (Tejero et al, 1996), CONGEN structures generated from extended starting conformations (with randomly assigned initial velocities) also exhibit fewer constraint violations and lower energies than structures generated using a hybrid DG/SA algorithm.…”

“…A detailed summary of considerations used in generating distance constraints involving degenerate protons from experimental NOE data is presented in the companion paper (Tejero et al, 1996). The parameter r,, is determined from the upper-bound of each distance constraint (rub), the weighting factor ( K N O E ) , and the maximum force (F,,,,) as follows:…”

“…In this "weight annealing" process (see the Methods), weights on the NMR constraint terms are increased, whereas weights on the conformational energy terms are scaled down, allowing the trajectories to evolve primarily under the influence of the NMR constraints. Weight annealing prior to the temperature annealing process was found to provide improved sampling of solution space and to yield structures at the end of the SA process that better satisfied simulated or real NMR constraints (see also Tejero et al, 1996).…”

Disulfide bond plasticity in epidermal growth factor

“…Biological data from EGF and at least one EGF-like domain show that disulfide bond isomers have significant bioactivity and suggests that the EGF fold can accommodate alternate disulfidebonding patterns. The disulfide bonds in murine EGF were altered to seven different patterns and structures were calculated incorporating all the restraints from the highest resolution restraint set available (Tejero et al, 1996). Results showed that besides the native (1-3,2-4,5-6), two other disulfide-bonding patterns: (1-2,3-4,5-6) and (1-3,2-5,4-6) satisfied the restraints as well as the native.…”