Abstract
Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.
Highlights
Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins
Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a, but that they are not fixed or long lasting
We have addressed the question of whether the ion pairs formed between specific residues in the myosin 7a (M7A) SAH are strong and persistent, whether the ion pairs change, and if ion pairs are even important for stability at all, which has been questioned
Summary
An initial characterization of the full-length M7A SAH (Fig. S1) by CD, analytical ultracentrifugation (AUC), size-exclusion chromatography, and small-angle X-ray scattering (SAXS) demonstrated that our M7A SAH construct (residues 858 – 935) exhibited the properties of previously well-characterized SAHs in solution [2, 6, 10, 16]. The H⑀–N⑀ peaks for central Arg residues show small positive heteronuclear NOEs (Fig. 4D and Table 1) consistent with the side chains being relatively ordered compared with those of the N and C termini Those located close to the N or C terminus have clear negative 1H–15N heteronuclear NOEs. Whereas the comparatively high values of the NOEs indicate a restricted or slower timescale of local motion, the values do not approach those measured for the backbone residues, nor those of Arg N⑀ involved in binding to phosphotyrosine [43]. This again is an indicator that central Arg side chains are more constrained than
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