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Assignment of side-chain conformation using adiabatic energy mapping, free energy perturbation, and molecular dynamic simulations

Published online by Cambridge University Press:  01 January 1999

THOMAS M. FRIMURER
Affiliation:
MedChem Research IV, Novo Nordisk Park, Novo Nordisk A/S, DK-2760 Måløv, Denmark
GÜNTHER H. PETERS
Affiliation:
Chemistry Department III, H.C. Ørsted Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
MORTEN DAHL SØRENSEN
Affiliation:
Chemistry Department V, H.C. Ørsted Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark Present address: Department of Chemical Research, Leo Pharmaceutical Products, DK-2750 Ballerup, Denmark.
JENS JØRGEN LED
Affiliation:
Chemistry Department V, H.C. Ørsted Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
OLE HVILSTED OLSEN
Affiliation:
MedChem Research IV, Novo Nordisk Park, Novo Nordisk A/S, DK-2760 Måløv, Denmark
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Abstract

NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (α3(VI)) from the human α3-chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X-ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X-ray crystallographic structure and solution structure of α3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free-energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77 ± 0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the α3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13–Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp21 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.

Type
Research Article
Copyright
© 1999 The Protein Society

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