Solution structure of paramagnetic metalloproteins

Abstract

Paramagnetism causes broadening of the NMR lines and therefore makes dif®cult the detection of the constraints (NOEs and 3 J) which are necessary for the determination of solution structures. The broadening is due to the fast nuclear relaxation rates, which are induced by the coupling of the nucleus with the unpaired electrons. Nevertheless, an NMR methodology has been developed, allowing the detection of classical constraints. This has allowed us to solve the ®rst solution structure of a paramagnetic metalloprotein in 1994. Since then, several solution structures of paramagnetic proteins have appeared. In addition, paramagnetism has been exploited in order to obtain new, nonclassical constraints. First, the nuclear relaxation has been exploited to obtain metal±proton distances. The position of nuclei with respect to the magnetic susceptibility tensor axes has been determined by the use of pseudocontact shifts. The contact shifts have been used as constraints after discovering or con®rming the shift dependence on dihedral angles. Paramagnetic molecules can be strongly magnetically anisotropic and therefore display partial orientation in strong external magnetic ®elds. These orientational effects result in dipolar contributions to the 15 N-1 H 1 J coupling. Such contributions can yield powerful structural constraints. In summary, the solution structure of many paramagnetic metalloproteins has been solved and described, and several new strategies have been developed for such solution structure determinations.

A Caged Lanthanide Complex as a Paramagnetic Shift Agent for Protein NMR

“…For some time, paramagnetism was considered a nuisance in NMR structure determination 1. 2 However, in recent years, it has been demonstrated that paramagnetism can provide useful structural information and, as a result, determination of the solution structure of proteins containing paramagnetic metals is now a reality 3. 4 The presence of an unpaired electron can cause both broadening and shifts of the resonances in the NMR spectrum.…”

“…(1)] in the Experimental Section) and falls off with the third power of the distance between the metal and the observed nucleus. For strongly paramagnetic metals with a high anisotropy, PCS can be observed for nuclei at long distances from the metal,5, 6 which can be used to provide restraints for structure determination 2. 4 Paramagnetic molecules with strongly anisotropic magnetic susceptibility also show some degree of spontaneous alignment at high magnetic fields; this provides an easy way to obtain residual dipolar couplings 7.…”

Nuclear Magnetic Resonance Shifts in Paramagnetic Metalloporphyrins and Metalloproteins

“…There has been intense interest in investigating the structures of metalloporphyrins and metalloproteins for many years. − For metalloporphyrins, X-ray methods give very accurate three-dimensional structural information, , but X-ray methods are less accurate for the larger protein structures. There is thus interest in using spectroscopic methods to investigate such systems.…”

Tuning the Affinity for Lanthanides of Calcium Binding Proteins

“…They are widely employed as luminescent probes (19,20) as well as NMR probes. In the latter case, their paramagnetism is exploited to generate new constraints in solution structure determinations (21)(22)(23)(24)(25)(26)(27). In view of these favorable properties, it would be desirable to have stoichiometrically definite and selectively substituted lanthanide-bound proteins (28).…”