Peter Krüger

Targeted molecular dynamics simulation of conformational change: application to the T↔R transition in insulin

Abstract A novel method to calculate transition pathways between two known protein conformations is presented. It is based on a molecular dynamics simulation starting from one conformational state as initial structure and using the other for a directing constraint. The method is exemplified with the T ↔ R transition of insulin. The most striking difference between these conformational states is that in T the 8 N-terminal residues of the B chain are arranged as an extended strand whereas in R they are forming a helix. Both the transition from T to R and from R to T were simulated. The method proves capable of finding a continuous pathway for each direction which are moderately different. The refolding processes are illustrated by a series of transient structures and pairs of O, ψ angles selected from the time course of the simulations. In the T → R direction the helix is formed in the →last third of the transition, while in the R → T direction it is preserved during more than half of the simulation period....

Targeted molecular dynamics: A new approach for searching pathways of conformational transitions

Molecular dynamics simulations have proven to be a valuable tool to investigate the dynamic behavior of stable macromolecules at finite temperatures. However, considerable conformational transitions take place during a simulation only accidentally or at exceptionally high temperatures far from the range of experimental conditions. Targeted molecular dynamics (TMD) is a method to induce a conformational change to a known target structure at ordinary temperature by applying a time-dependent, purely geometrical constraint. The transition is enforced independently of the height of energy barriers, while the dynamics of the molecule is only minimally influenced by the constraint. Simulations of decaalanine and insulin show the ability of the method to explore the configurational space for pathways accessible at a given temperature. The transitions studied at insulin comprise unfolding of an alpha-helical portion and, in the reverse direction, refolding from an extended conformation. A possible application of TMD is the search for energy barriers and stable intermediates from rather local changes up to protein denaturation.

A COMPARISON OF THE STRUCTURE AND DYNAMICS OF AVIAN PANCREATIC-POLYPEPTIDE HORMONE IN SOLUTION AND IN THE CRYSTAL

A molecular dynamics simulation was carried out with avian pancreatic polypeptide hormone (aPP) as an isolated monomer explicitly including the solvent (MDS). The simulation and the resulting mean structure are compared with the results of a corresponding crystal simulation (MDC) with 4 aPP molecules plus interstitial water in a periodic boundary unit cell and with the X-ray structure (van Gunsteren, Haneef et al., manuscript in preparation). Comparison is based on the time span 5 to 15 ps and considering cartesian coordinates, dihedral angles, H-bond length, and accessible surface area. While in the MDC simulation equilibration is fast and complete, it does occur in MDS for most but not all parts of the molecule; the turn region starts moving away from the X-ray structure after 9 ps.Only minor differences result when dimerforming side chains, e.g. tyrosines 7 and 21, are exposed to solvent. The largest rms fluctuations are encountered in exposed polar side chains of Asp 11, Glu 15, Arg 19, and Arg 33, but also in the hydrophobic core residue Phe 20, the only phenylalanine residue present. The latter undergoes an abrupt reorientation suitable for verification by NMR spectroscopy, which is possibly related to the motion of the turn region. The main-chain dihedral angles of the α-helix are shifted from values generally found in crystal structures towards those of the ideal Pauling helix. There is concomitant H-bond elongation. The effects are most pronounced and consistent in the MDS simulation.

The simulated dynamics of the insulin monomer and their relationship to the molecule's structure

Insulin crystallizes in different forms, some of which show different conformations for the different molecules in the asymmetric unit. This observation leads to the question as to which conformation the molecule will adopt in solution. Molecular dynamics computer simulations of rhombohedral 2 Zn pig insulin have been carried out for both monomers (1 and 2) independently in order to study their behaviour in the absence of quaternary structure and crystal packing forces.These preliminary 120 ps simulations suggest that both monomers converge in solution to very similar conformations which differ from the X-ray structures of both monomer 1 and 2 (Chinese nomenclature), but are closer to the former, as has previously been suggested by an analysis of the crystal packing (Chothia et al. 1983) and by energy minimization (Wodak et al. 1984). The secondary structure of the molecules is basically preserved, as expected. A detailed description of the conformational changes is given.

CALCULATION OF THE CIRCULAR DICHROISM SPECTRUM OF CYCLO-(L-TYR-L-TYR) BASED ON A MOLECULAR DYNAMICS SIMULATION

Theoretical calculations of CD spectra have generally assumed a single conformation, or a small number of conformers with Boltzmann averaging. Solvent effects on both the conformation and the CD have been neglected. In this work, we have calculated the CD spectrum of cyclo(L-Tyr-L-Tyr) in aqueous solution, taking dynamics and solvation into account. Starting geometries with chi 1 approximately 300 degrees or 60 degrees for both Tyr side chains were derived from MNDO/MOPAC, followed by energy minimization using GROMOS. After addition of 368 water molecules, the system was simulated for 1000 ps at 300 K using GROMOS. In addition to the starting conformer, two other conformers were observed during each simulation. However, each trajectory gave a distinct set of conformers. Rotational strengths were calculated for the cyclic dipeptide at each ps along the trajectories, using the matrix method. The CD spectra calculated from these rotational strengths were averaged over the trajectories. Agreement is very good for the strong negative band near 200 nm, while for the lower energy bands (near 230 and 280 nm), the signs are correct, but the magnitudes are too low. The spectrum calculated from a Boltzmann-weighted average over the in vacuo MNDO/MOPAC conformers was in poor agreement with experiment. Although the solvent did not significantly affect the rotational strength calculated for a given conformer, it is essential to include the solvent in the MD simulations because it affects the relative energies of the conformers and promotes transitions among them.

Modeling of G-protein coupled receptors with bacteriorhodopsin as a template. A novel approach based on interaction energy differences

The structure of bacteriorhodopsin was used as a template to generate a model for G-protein coupled receptors. However, these receptors and the template are not related by sequence homology. Therefore a pragmatic and reproducible approach was developed to achieve an energetically favourable accommodation of receptor sequences to the backbone structure of bacteriorhodopsin. Improved interaction energy differences are used in a two step procedure analogous to a hypothetical folding mechanism for integral membrane proteins. The resulting model is in good agreement with existing data from structure-function studies.

MD simulation of protein-ligand interaction: formation and dissociation of an insulin-phenol complex.

Complexes of proteins with small ligands are of utmost importance in biochemistry, and therefore equilibria, formation, and decay have been investigated extensively by means of biochemical and biophysical methods. Theoretical studies of the molecular dynamics of such systems in solution are restricted to 10 ns, i.e., to fast processes. Only recently new theoretical methods have been developed not to observe the process in real time, but to explore its pathway(s) through the energy landscape. From the profiles of free energy, equilibrium and kinetic quantities can be determined using transition-state theory. This study is dedicated to the pharmacologically relevant insulin-phenol complex. The distance of the center of mass chosen as a reaction coordinate allows a reasonable description over most of the pathway. The analysis is facilitated by analytical expressions we recently derived for distance-type reaction coordinates. Only the sudden onset of rotations at the very release of the ligand cannot be parameterized by a distance. They obviously require a particular treatment. Like a preliminary study on a peptide, the present case emphasizes the contribution of internal friction inside a protein, which can be computed from simulation data. The calculated equilibrium constant and the friction-corrected rates agree well with experimental data.

Comparative studies on the dynamics of crosslinked insulin

Molecular dynamics simulations were carried out on an insulin crosslinked between the N-terminal A chain and the C-terminal B chain to form a so-called mini-proinsulin: Nα-A1-Nε-B29-diaminosuberoyl insulin (DASI). To investigate the influence of crosslinking on the dynamics of the insulin moiety, the bridge was removed from a transient DASI structure and simulation was carried on independently with the then unlinked (ULKI) as well as with the crosslinked species. The effects of crystal packing and quaternary interactions were checked by simulating both types of monomers and dimers known from the hexamer structure. All simulations were compared to previous ones of native insulin. DASI shows general similarity to the native simulations in most parts of the structure. Deviations are visible in the segments to which the bridge is directly connected, i.e. their flexibility is reduced. Upon removal of the bridge the ULKI simulations reapproach those of native insulin. The influence of the bridge spreads over the whole molecule, but all of its main structural features remain intact. The simulations suggest that the displacement of the C-terminal B chain of native insulin, considered important for receptor interaction, is prevented by the bridge, which also partially shields some binding residues. This is in accordance with the poor biological potency of A1-B29-crosslinked insulins.

SIMLYS version 2.0

Abstract SIMLYS is a tool to analyze the results of molecular simulations. It provides functions to investigate the coordinates and in addition it helps to generate and integrate new analysis functions. The version 2.0 of the program contains a number of new modules, as for instance the calculation of accessible surface area, nearest neighbours and correlation functions. The facility to generate a new analysis subroutine was extended and it was made possible to integrate the new module into the present program background. A number of existing modules were updated and the command interpreter was improved.