Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix–to–β-strand and β-strand–to–α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.
Solution-state NMR can be used to study protein–lipid
interactions,
in particular, the effect that proteins have on lipids. One drawback
is that only small assemblies can be studied, and therefore, fast-tumbling
bicelles are commonly used. Bicelles contain a lipid bilayer that
is solubilized by detergents. A complication is that they are only
stable at high concentrations, exceeding the CMC of the detergent.
This issue has previously been addressed by introducing a detergent
(Cyclosfos-6) with a substantially lower CMC. Here, we developed a
set of bicelles using this detergent for studies of membrane-associated
mycobacterial proteins, for example, PimA, a key enzyme for bacterial
growth. To mimic the lipid composition of mycobacterial membranes,
PI, PG, and PC lipids were used. Diffusion NMR was used to characterize
the bicelles, and spin relaxation was used to measure the dynamic
properties of the lipids. The results suggest that bicelles are formed,
although they are smaller than “conventional” bicelles.
Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing
PI and PC. The paramagnetic label was shown to have a shallow location
in the bicelle, affecting the glycerol backbone of the lipids. We
foresee that these bicelles will be useful for detailed studies of
protein–lipid interactions.
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