Chemistry Reference
In-Depth Information
1.4 Paramagnetic NMR
As NMR spectroscopists are constantly on the quest to improve the line-shape and to
reduce the width of the peaks in their spectra, elimination of the paramagnetic
species, often causing significant line-broadening effects, has been considered as
paramount in sample preparations. However, the usually undesirable line-broadening
effect can provide unique structural long range information [ 29 ] when the effect is
specific and paramagnetic center is localized to a particular site of the
macromolecules. Historically, this understanding has been mainly applied to proteins
containing metal-binding sites (reviewed in [ 30 ]). The idea to utilize surface exposed
cysteines for introduction of paramagnetic tags came later with a cornucopia of
chemical agents and procedures developed for reliable conjugation at specific sites
(reviewed in [ 31 ]). This approach not only provides information about intramolecular
distances but can also help in defining alignment of binding subunits within
complexes characterized by wPPIs [ 32 ], although actual quantification of the distance
dependence in such systems is not always straightforward as we discuss below. Two
distinguished NMR phenomena, based upon the specific nature of the magnetic
moment of an attached paramagnetic center, present the basis for structural investi-
gation. These are paramagnetic relaxation enhancement (PRE) characteristic for all
paramagnetic moieties and the pseudocontact shifts (PCS) effect specific for the
subclass of paramagnetic ions with an anisotropic electron g -tensor.
1.4.1 The PRE Effect
The large magnetic dipolar interaction of the unpaired electron from a paramagnetic
atom with the neighboring NMR-active nucleus results in an increase of the relaxa-
tion rate of the above nucleus [ 33 ]. Similar to NOE, this effect has basic r 6 distance
proportionality, but, because of the larger magnetic moment of the electron, PRE
effect is observable at longer, up to 25-35 ˚ , distances depending upon the nature of
the particular paramagnetic group [ 34 , 35 ]. Thus PRE measurements can provide
much longer range distance restraints for structural calculations in comparison to the
classical NOE approach. The caveat of PRE application for short distances determi-
nation comes from the same original source and relates to the fact that nuclei situated
very close to a paramagnetic center are often broadened beyond detection. However,
the data sets acquired by NOE and PRE approaches are at least complementary. The
PRE measurements are based on the correlation of the increased transverse relaxation
rate with the distance between the introduced paramagnetic moiety and the affected
nucleus [ 36 ]. Simply speaking, we are measuring the distance-dependent reduction in
peak intensities in a 15 N-HSQC spectrum of the target protein when a single
paramagnetic tag is attached to it at the specific site, usually through a thioether
bond formed with the side chain of the cysteine residue. Nitroxide stable radicals or
metal chelators, such as EDTA-Mn 2+ , which are characterized by an unpaired
electron with an isotropic g-tensor, are especially useful since they do not give rise
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