Supplementary Results
to
Electrostatic couplings in OmpA ion channel gating suggest a
mechanism for pore opening
by
Heedeok Hong, Gabor Szabo and Lukas K. Tamm
Duplication of double mutant cycles with different sets of mutants. Several
interactions were measured with two independent double mutant cycles (Figure 3 in main
text). A change of Glu52 to an Ala gave the same interaction energy as the more
conservative change to a Gln in both the Glu52–Arg138 and Glu52–Lys82 interaction.
Similarly, the two double mutant cycles with Glu128Ala and Glu128Gln
replacements that probed the Glu128−Arg138 interaction yielded essentially the same
values. We also attempted the duplicate measurement of the Tyr8–Glu52 interaction by
replacing Glu52 with an Ala instead of a Gln. However, we encountered difficulties with
quantitatively refolding the Y8A/E52A double mutant. Contrary to all other mutants
presented in this work, which refolded to 90-100%, this particular mutant refolded to
only ~70%. Stability measurements of this mutant therefore yielded only lower limits,
which however resulted in an interaction energy by mutant cycle analysis that was still
consistent with the energy measured with the Gln-containing mutant cycle.
Analysis of m-values in double mutant cycles. As shown in Table 1, the m-values are
broadly distributed over a range of 1.8~3.1 kcal/mol M-1. Repeated unfolding
measurements of each protein indicate that this distribution is real and does not originate
from experimental errors. The experimental m-values of many proteins have been
correlated with the hydrophobic surface area that is exposed upon unfolding1. For a set
of mutant proteins with approximately the same native structures, the variation of mvalues may be attributed to changes in the denatured state ensembles rather than to
structural perturbations of the folded state2. Double mutant cycle analysis assumes that
the folded and denatured states are not grossly perturbed and that the interacting pairs of
the folded state should not interact in the denatured state. Therefore, one might suspect
that the broad m-value variation observed in this study could be affected by perturbations
of the native or denatured states. However, a thorough analysis of the m-value changes in
each double mutant cycle shows that the m-value effects are additive and effectively
cancel out. For example, the m-value changes by m=−0.4 from wild-type to K92A
mutant. The same change (m=−0.4) is observed in the parallel step from E52Q to
E52Q/K82A in the double mutant cycle (Fig. 3 and Table 1). The parameter that
describes the non-additivity of m-values for single and the corresponding double mutants
is defined as
m=mXY-00-(mXY-X0+mXY-0Y),
(S1)
1
where mXY-00 designates a difference in m-value between wild-type and a double mutant,
and mXY-X0 and mXY-0Y corresponds to the difference between wild-type and each single
mutant3. In our eight double mutant cycles, the m’s vary in a narrow range from −0.2
to 0.4 (Supplementary Fig. 7). We estimate the uncertainty in m to be <±0.2 from
propagation of the errors of the individual m-values. Therefore, the changes in m-values
are approximately additive, implying that the energies measured from our double mutant
cycles indeed represent the pairwise side-chain interaction energies. This notion is
further supported by the observation that the same interacting energies are obtained when
the same residue pairs are probed with different mutant cycles (Fig. 3). In addition, all
residues whose interactions are probed in this work are far apart from each other in
sequence, and therefore are unlikely to interact in the denatured states.
References
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Myers, J.K., Pace, C.N. & Scholtz, J.M. Denaturant m values and heat capacity
changes: relation to changes in accessible surface areas of protein unfolding
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Shortle, D. Staphylococcal nuclease: a showcase of m-value effects. Adv Protein
Chem 46, 217-47 (1995).
Green, S.M. & Shortle, D. Patterns of nonadditivity between pairs of stability
mutations in staphylococcal nuclease. Biochemistry 32, 10131-9 (1993).
2