Chemistry Reference
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calculated for Mn 2+ -EDTA labeled DNA in complex with SRY. This type of
ensemble-based refinement has also been suggested to be necessary for more
accurate use of PRE data from the MTLS spin label, particularly since some
high-resolution crystal structures of MTSL-modified lysozyme show the presence
of more than one spin-label conformer [ 369 , 370 ]. However, the good agreement
that was found between calculated and PRE-measured distances for the relatively
rigid barrel region of MTSL-labeled OmpA implies that the presence of multiple
conformations may be less significant for this particular spin label [ 367 ]. EPR line
shapes of MTSL-modified lysozyme also show that the motion of this paramagnetic
center is restricted [ 371 ]. Hence it appears that carefully measured PREs with
MTSL in non-dynamic segments can in most cases be validly converted into
long-range distance restraints with tighter bounds (i.e.,
2 ˚ ).
Overall, PREs have significantly increased the number of larger membrane
protein structures that have been determined by solution NMR in a relatively
short period, becoming an established source of structural information. In addition,
water-soluble paramagnetic relaxation agents can be used to reveal aqueous-
exposed segments [ 136 , 162 , 184 , 354 , 366 , 372 - 374 ], spin-labeled lipids and
detergents can identify lipid-embedded regions [ 136 , 372 , 375 - 381 ], and intermo-
lecular interactions can be probed with exquisite sensitivity using spin-labeled
proteins [ 243 , 382 ]. This versatility is one of the factors ensuring that paramagnetic
effects in NMR will continue to make important contributions to our understanding
of membrane protein structure.
6 Concluding Remarks
Although membrane proteins continue to present challenges for solution NMR,
innovations in sample development, data acquisition, and structure determination
strategies have allowed structural insights to be obtained from some of the most
demanding systems tackled to date (e.g., GPCRs and large
-barrel channels). In
addition to the advances outlined here, these achievements build upon contributions
from the handful of veteran laboratories that have worked to apply solution NMR to
membrane proteins, even before the introduction of more modern methods for study
of large proteins (e.g., [ 140 , 383 - 386 ]). Yet in spite of this long history, the field is
still considered to be quite young, with only a limited number of groups fully
exploiting the potential of solution NMR for membrane protein structure determi-
nation. Nonetheless, it is encouraging to note the progress that has been made since
the first NMR structure was determined for an integral membrane protein
comprised of more than one TM segment back in 1997 (i.e., the glycophorin A
homodimer [ 140 ]). As of the end of 2010 the protein structure database holds
approximately 24 integral membrane protein structures (counting only those with
more than one membrane-spanning segment), putting the field at par with the state
of membrane protein crystallography in the late 1990s. However, the rate of new
structure accumulation closely follows the exponential rise seen for crystal
b
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