Methods for Studying the Interactions of Translation Factors with the Ribosome - Biophysical Approaches to Translational Control of Gene Expression - page 89

Fig. 5.1 Directed hydroxyl radical cleavages of 18S rRNA from specific positions of human
eIF5B. The rRNA is shown as beige ribbon and ribosomal proteins are shown in surface represen-
tation. The docked model of eIF5B is shown in purple ribbon. The positions of cysteines on the
surface of domain 2 of eIF5B are shown as colored spheres with asterisks . The cleavage positions
in 18S rRNA are shown as spheres with the same color as that of the cysteine from which they are
cleaved, and the radii of the spheres correspond to the cleavage intensities: weak, medium, and
strong. ( a ) Cleavages from C884 ( red ) and C894 ( violet ). ( b ) Cleavages from C952 ( cyan ) and
C961 ( blue ). The distances between several cleavage positions and corresponding eIF5B residues
are shown. Some of the helices in rRNA are labeled, e.g., h3 is helix 3. eIF5B domain 2, where the
above cysteines are located is labeled with “D2.” Note that the pairs of mutants shown in panels
( a ) and ( b ) produce nonoverlapping sets of cleavages in rRNA, including strong cleavages of
nucleotides expected to lie as far as ~50 Å away. Since the distances between C884 and C894 and
between C952 and C961 are only ~27 Å and ~10 Å, respectively, and the length of the Fe(II)-
BABE linker itself is ~10 Å, these cleavage patterns indicate that: (1) the Fe(II)-BABE linker is not
moving freely in the ribosomal complexes; and (2) the cleaved rRNA segments that appear distant
in the docking model are likely brought closer to eIF5B in the complex through conformational
changes. Reproduced from Unbehaun et al. ( 2007 ) with permission

5.5.3 Cysteine-Less vs. Partially Cysteine-Less Mutants

It is often impossible to mutate all existing cysteines in the protein of interest,
because the resulting cysteine-less mutant protein is not active and/or unstable. In
most cases, that is not a problem, as long as derivatization with Fe(II)-BABE itself
does not inactivate the protein. A partially cysteine-less protein can be used, instead
by mutating only those cysteines that are not essential for structure or function.
Cysteines that are important for structure and stability are usually buried and not
accessible for derivatization. Even if some of the endogenous cysteines in a partially
cysteine-less mutant do get modified, that would not invalidate the results, because
any cleavages from such cysteines would be present in the negative control (an
Fe(II)-BABE-treated partially cysteine-less mutant) as well as in all complexes with
individual cysteine mutants.