Supplementary data
Solution structure of lysine-free (K0) ubiquitin
Tao Huang, Jess Li and R. Andrew Byrd*
Structural Biophysics Laboratory,
Center for Cancer Research
National Cancer Institute, Frederick, MD
Figure S1. Assigned two-dimensional 1H-15N HSQC spectrum of K0-Ub.
A
talos_phi K0-Ub vs wt-Ub
100
Phi (K0-Ub)
50
0
-50
-100
-150
-150
-100
-50
0
50
100
150
200
Phi (wt-Ub)
B
talos_psi K0-Ub vs wt-Ub
200
Psi (K0-Ub)
150
100
50
0
-50
-50
0
50
100
Psi (wt-Ub)
Figure S2. Plots of Phi (A) and Psi (B) angles of K0-Ub versus wt-Ub from TALOS calculation. One
outlier on the psi plot was D52, which is involved in a salt bridged with K27 (Table S1).
Figure S3. Thermal-induced unfolding curves of ubiquitin (Tm = 90.6 °C) and K0-Ub (Tm = 71.9 °C)
measured by DSC.
Table S1. Hydrogen bonding and salt-bridge interactions involving Lys NH3+ groups in wt-Ub (PDB:
1UBQ)
Donor
Acceptor
Distance (Å)
K11 Nζ
E34 Oε
3.347
K27 Nζ
Q41 O
3.245
K27 Nζ
D52 Oδ
2.902
K29 Nζ
E16 O
2.676
K33 Nζ
T14 O
3.407
K48 Nζ
A46 O
3.379
Concerning the issue of minor species in the spectra of K0-Ub:
Anecdotally, there are differing amounts of minor conformation observed from different preparations of
K0 ubiquitin, but the smallest minor populations always occur when the protein is purified from only the
soluble fraction. When the protein is refolded, differing (and usually larger) amounts of minor species
are observed. Since ubiquitin is so evolutionarily conserved and stable, consistent with its role as a posttranslational modifier, it is likely that the form purified from the soluble fraction following expression
represents the low energy form. Consequently, we feel that the structure reported represents this form
and has been shown to be equivalent to wild-type ubiquitin.
When a significant number of alterations affecting H-bonds and salt-bridges occur throughout the
protein, it is possible to trap minor populations with varying orientations of some of these components.
The data suggests that there is not a single population for the minor species, and it is most likely that the
ensemble of molecules represents cases where the major conformation exists for most of the residues
and a small number of residues in any given molecule exhibit a different conformation or perturbation of
an H-bond or salt-bridge. In this situation, there can be different percentages of minor conformations at
different residues without presupposing that there is a single major species and a single minor species.
Thus, the most stable conformation is represented by the major peaks in the spectrum, and the minor
peaks represent low populations of alternate conformations, which may exist in isolated segments within
the ensemble, rather than a single alternate conformation. This view is supported both by the nature of
the perturbations and the fact that there is a range of populations observed across the residues in
Ubiquitin. The population of “assignable” minor species ranges from 4% to 26%.
We have prepared a plot of the CSP for the minor species relative to the wild-type Ubiquitin. This
shows that there are perturbations distributed through the structure. The scale of the perturbations is
smaller than for the major species, but not disparately so. This plot is somewhat misleading, as it does
not express the population of the species/peaks. We can assign peaks in triple resonance spectra for 27
residues, with populations ranging from 4% to 26% (indicated in the CSP plot by red bars, referenced to
the right vertical axis). A very interesting aspect of these assignments is that it is impossible to
unambiguously assign the minor peaks to a single molecular species. First, the population variation
suggests that there is not a single molecular species representing the minor conformation. Second, the
triple resonance spectra exhibit degeneracy for the Ca, Cb, and C’ shifts, hence, it is impossible to trace
a connectivity through only the minor peaks. Instead, it is more likely that the minor conformations are
distributed with perhaps one or a few alternate forms in any single molecular species that is, otherwise,
predominantly the major form. The rate of exchange is clearly slow on the NMR timescale, and, based
on the chemical shift separation (as small as 0.015 ppm [15N]), the rate is slower than 1.05 sec-1. Careful
examination of the minor resonances, which are populated at more than 10%, suggests that the chemical
shifts are most likely due to local perturbations in a side-chain to side-chain hydrogen bond formed by a
Lys sidechain that is mutated to Arg in K0-Ub or a salt-bridge. Replacement of the Lys ξ-amino group
with the Arg guanadino moiety could lead to either loss of the hydrogen bond or compensation to pack
in the bulkier sidechain and the possibility of two inequivalent rotamers of the Arg sidechain enabling
the formation of the hydrogen bonds via either the η1 or η2 amino groups. Indeed, some of the
perturbations are correlated with an approximately equal population, such as those of I13 and L15 with
R33 and R29 (these are R in the K0-Ub).
Based on this analysis of the data from the minor species, it is clear that the principal tenet of our
manuscript is maintained, namely that the three-dimensional structure of K0-Ub is equivalent to wtubiquitin. While intriguing and potentially the subject of subsequent detailed investigation, which could
contribute to the understanding of protein folding and stability, we respectfully submit that this goes
beyond the nature of this short communication of a valuable new protein structure. The stability of the
K0-Ub fold, as well as the equivalence for the fold of the minor conformers (based on the Ca, Cb, C’
shifts and the respective torsion angles from TALOS), suggests that K0-Ub is indeed a viable molecular
replacement for wt-ubiquitin in biochemical and cellular studies, where the ability to control
ubiquitination and chain extension is desired.