Maximum Occurrence

Flexible proteins and protein complexes may sample a wide conformational space. In these cases, pseudocontact shifts and paramagnetism-based residual dipolar couplings provide experimental observables averaged over many conformation with different weights. The Maximum Occurrence is the maximum percent of time that a conformation of a macromolecule can exist and still be compatible with the experimental data. Recently, an implementation of this method based on distributed computing was presented to evaluate the Maximum Occurrence profiles for a large number of conformers.

350px-MO_profilesThe Maximum Occurrence of a conformation of a protein is determined from the increase in the target function with increasing the weight of the conformation within the ensemble.


Fragai, Luchinat, Parigi, Ravera, Conformational freedom of metalloproteins revealed by paramagnetism-assisted NMR, Coord Chem Rev 257, 2652-2667, 2013.

Cerofolini, Fields, Fragai, Geraldes, Luchinat, Parigi, Ravera, Svergun, Teixeira, Examination of matrix metalloproteinase-1 (MMP-1) in solution: a preference for the pre-collagenolysis state, J Biol Chem 288, 30659-30671, 2013.

Bertini, Giachetti, Luchinat, Parigi, Petoukhov, Pierattelli, Ravera, Svergun, Conformational space of flexible biological macromolecules from average data, J Am Chem Soc, 132, 13553-13558, 2010.

Bertini, Gupta, Luchinat, Parigi, Peana, Sgheri, Yuan, Paramagnetism-based NMR restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains. J Am Chem Soc, 129, 12786-12794, 2007

Paramagnetism Based Restraints Implemented in CYANA and Xplor-NIH

Classical protein structure determination by NMR relies on short-range proton-proton distances (NOEs). Despite successful application to the study of compact, globular molecules, this method encounters severe limitations when applied to larger or more complex systems. In particular, the presence of one of more paramagnetic centers in a protein produces a large spread in the chemical shifts and an increase of the longitudinal and transverse relaxation rates. The latter effect causes an increase in the threshold for detection of cross-peaks between nuclei, which may be important for nuclei close to the paramagnetic center, with the consequence that the weakest cross-peaks can be lost in the noise. Furthermore, no information is usually available a priori on the position of the metal ion. On the other hand, experimental measurements of several effects due to the paramagnetic metal ion, such as contributions to chemical shifts, can provide important structural information. It is thus critical to the structural determination of paramagnetic metalloproteins that such information is properly exploited. This goal can be achieved through the inclusion of novel restraints within the available computational methods for structure determination. To date, most NMR structures of paramagnetic proteins have been solved using the program CYANA, which implements a simulated annealing procedure with molecular dynamics in torsion angle space (Guntert, 1997), or Xplor-NIH (Clore, 1985; Schwieters, 2003). The capabilities of CYANA have been extended by the addition of new modules which allow the use of pseudocontact shifts, paramagnetic residual dipolar couplings and cross correlation rates as structural restraints. These features are collected in PARAMAGNETIC-CYANA. A complete set of modules for paramagnetism-based restraints was also integrated in Xplor-NIH under the collective name of PARArestraints for Xplor-NIH.

I. Bertini, P. Kursula, C. Luchinat, G. Parigi, J. Vahokoski, M. Wilmanns, J. Yuan, 2009, “Accurate solution structures of proteins from X-ray data and a minimal set of NMR data: calmodulin-peptide complexes as examples”, J. Am. Chem. Soc. 131, 5134–5144.
S. Balayssac, I. Bertini, C. Luchinat, G. Parigi, M. Piccioli, 2006, “13C direct detected NMR increases the detectability of residual dipolar couplings”, J. Am. Chem. Soc. 128, 15042-15043.
Bertini I, Luchinat C, Parigi G, Pierattelli R, 2005, "NMR Spectroscopy of Paramagnetic Metalloproteins", ChemBioChem 6:1536-1549. 
Banci L, Bertini I, Cavallaro G, Giachetti A, Luchinat C, Parigi G, 2004, "Paramagnetism-based restraints for Xplor-NIH", J. Biomol. NMR 28:249-261. 
Barbieri R, Luchinat C, Parigi G, 2004, "Backbone-only protein solution structures with a combination of classical and paramagnetism-based constraints: a method that can be scaled to large molecules", Chem Phys Chem 5:797-806. 
Bertini I, Luchinat C, Parigi G, 2002, "Paramagnetic constraints: an aid for quick solution structure determination of paramagnetic metalloproteins", Concepts in Magn Reson 14:259-286. 
I. Bertini, C. Luchinat, G. Parigi, "Magnetic susceptibility in paramagnetic NMR", Progr. in NMR Spectr. (2002) 40, 249-273. 
I. Bertini, C. Luchinat, M. Piccioli, “ Paramagnetic probes in metalloproteins”, Methods Enzymol. (2001) 339, 314-340
Guntert, P, Mumenthaler, C, Wuthrich, K, “Torsion angle dynamics for NMR structure calculation with the new program DYANA”, J.Mol.Biol (1997) 273, 283-298
Clore, GM, Gronenborn, AM, Brunger, AT, Karplus, M, “Solution conformation of a heptadecapeptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli. Combined use of 1H nuclear magnetic resonance and restrained molecular dynamics”, J.Mol.Biol. (1985) 186, 433-455
Schwieters, CD, Kuszewski,JJ, Tjandra, N, Clore, GM, “The Xplor-NIH NMR molecular structure determination package”, J.Magn.Reson. (2003) 160, 65-73