Conformation of 3'CMP bound to RNase A using TrNOESY.
The conditions for accurately determining distance constraints from TrNOESY data on a small ligand (3'CMP) bound to a small protein (RNase A, <14 kDa) are described. For small proteins, normal TrNOESY conditions of 10:1 ligand:protein or greater can lead to inaccurate structures for the ligand-bound conformation due to the contribution of the free ligand to the TrNOESY signals. By using two ligand:protein ratios (2:1 and 5:1), which give the same distance constraints, a conformation of 3'CMP bound to RNase A was determined (glycosidic torsion angle, chi=-166 degrees ; pseudorotational phase angle, 0 degrees < or = P < or =36 degrees ). Ligand-protein NOESY cross peaks were also observed and used to dock 3'CMP into the binding pocket of the apo-protein (7rsa). After energy minimization, the conformation of the 3'CMP:RNase A complex was similar to the X-ray structure (1 rpf) except that a C3'-endo conformation for the ribose ring (rather than C2'-exo conformation) was found in the TrNOESY structure.
Lee
Yi-Chien
YC
National Cancer Institute at Frederick, Laboratory of Medical Chemistry, 376 Boyles Street, Building 376, Frederick, MD 21702, USA.
Jackson
Patricia L
PL
Jablonsky
Michael J
MJ
Muccio
Donald D
DD
D003198:Computer Simulation; D003568:Cytidine Monophosphate / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate