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the biggest membrane-protein structure solved by NMR. A member mitochondrial
anion carrier protein family, UCP2 translocates protons across the inner mitochon-
drial membrane. In order to determine the structure of UCP2, the newly developed
method needed to incorporate an effective way to circumvent traditional problems in
solving helical membrane-protein structures by NMR (discussed above), and allow
us to at least obtain information on the backbone structures. To accomplish this, the
new method combines the use of RDC and paramagnetic-relaxation-enhancement
(PRE) data.
This method consists of two steps:
Step 1: Determining local structural segments by RDC-based molecular fragment
replacement (MFR). As seen in previous examples, UCP2 exhibits severe signal
overlap that precludes it from NOE-based structure determination. To overcome
this, the DNA-nanotube liquid crystal medium was used to measure RDCs for
internuclear vectors 1 H N - 15 N( 1 D NH ), 13 C ' - 13 C ' ( 1 D C 0 C' ), and 15 N- 13 C 0 ( 1 D NC 0 ).
On average, there were 2.2 RDCs per residue for regions with confirmed resonance
assignment, which is shy of the three or more RDCs per residue typically required
to solve structures due to intrinsic orientation degeneracy of the dipolar-coupling
function. However, the new approach overcomes this by adapting the commonly
known molecular fragment replacement (MFR) method previously used for crystal-
lography. This method exhaustively searches the Protein Data Bank for fragments
of structures that agree with the RDCs from experimental data. By comparing a
database of 320,000 fragments from the Protein Data Bank with the RDCs data,
it was possible to identify 15 matching segments, covering most of the UCP2
sequence.
Step 2: Spatial arrangement of the MFR-derived segments to derive the tertiary
structure.
To determine the spatial arrangement of the MFR-derived segments in the tertiary
structure, semiquantitative distance restraints from paramagnetic-relaxation-
enhancement measurements (PRE) were used. PRE measurements provide
long-range distance information in the range of 15-24 A between a nitroxide
spin label covalently attached to cysteines (paramagnetic moiety) and NMR active
nuclei. For UCP2, a total of 452 PRE restraints have been measured using four
different nitroxide-labeled samples. Each sample was labeled with a spin label
attached at a unique position.
Finally, a two-stage procedure was used in the structure calculations. First, the
MFR fragments from the RDCs analysis were used to calculate local coordinates.
Second, the PRE restraints were introduced to determine the spatial arrangement of
the fragments. At the end, the calculations resulted in a structure ensemble with a
backbone precision of 1.3 ˚ A(Fig. 16.4 b).
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