Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins

Abstract

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed.

Solution Structure of Oxidized Horse Heart Cytochrome c,

“…In this way the position of the metal ion is defined by experimental constraints only. This calculation was performed through the program FAN-TASIA Banci et al, 1997c). In order to evaluate the discrepancies between calculated and experimental pseudocontact shifts, a target function (TF) is defined following the definition of the target function relative to the NOEs of the DYANA program: where the summation spans over all pseudocontact shift constraints, w i is the weight of the ith constraint (all taken equal in this work), δ exp pc is the ith measured pseudocontact shift, δ calc pc is the corresponding calculated pseudocontact shift, toll is the tolerance given to the pseudocontact shift constraints.…”

“…Pseudocontact shifts were included as constraints by means of a modified Sander module (PSEUDOREM) (Banci et al, 1997c).…”

800 MHz 1H NMR solution structure refinement of oxidized cytochrome c7 from Desulfuromonas acetoxidans

“…The distance limits between the Nε2 of the axial histidines and the iron ion were imposed by analogy with all the structures of heme proteins available in the Protein Data Bank [15Ϫ21]. The choice of the specific 'distal' ligand for each heme is based on the sequence alignment of D. acetoxidans cyt c 7 and Desulfovibrio cytochrome c3 [14] and was further checked through calculations performed without the introduction of any constraints between the iron ions and their axial ligands. When the upper distance limits involving the histidines' Nε2 and the iron ions were set equal to infinite the protein fold was the same, beside an increase in disorder of the imidazole rings.…”

“…The calculation was performed with the program FANTA-SIAN [7,9] which determines the above parameters fitting the experimental pseudocontact shifts simultaneously on all the structures of a family. The backbone atoms of the latter structures were first best superimposed.…”

“…The heme iron ions are bound to histidine residues from analogy with cytochrome c 3 from Desulfovibrio organisms, which have four hemes and which show large similarity with the present system [14Ϫ21]. The function of cyt c 7 has not yet been elucidated; however, it has been reported that it could function as a terminal reductase [22] and more recently that it acts as a polysulfide reductase [23]. It could represent the terminal reductase in D. acetoxidans.…”