Synthetic Multidomain Proteins Containing Unstructured and α‑Helical Linkers Reveal a Differential Impact of Molecular Crowding on Catalytic Activity and Conformation

Molecular crowding has been shown to impact both enzymatic
activity
and protein conformation individually. However, a simultaneous assessment
of its effect on the two parameters, especially in multidomain proteins,
has not yet been reported. Here, utilizing multidomain proteins containing
the mNeonGreen (mNG) fluorescent protein and the NanoLuc (NLuc) luciferase
fused by either unstructured or α-helical linkers of different
lengths, we report a differential impact of molecular crowding on
the enzymatic activity and protein conformation in a linker and crowder
size-dependent manner. Specifically, Gaussian accelerated molecular
dynamics (GaMD) simulations with representative unstructured and α-helical
linkers revealed differences in their structural dynamics. Simultaneous
monitoring of enzymatic activity through NLuc bioluminescence and
protein conformation through Bioluminescence Resonance Energy Transfer
between NLuc (donor) and mNG (acceptor) revealed both polyethylene
glycol molecular weight-dependent and linker length-dependent impacts
on NLuc enzymatic activity and conformation of an unstructured linker-containing
multidomain protein. Further, multidomain proteins containing α-helical
linkers of different lengths revealed a pronounced impact of molecular
crowding on NLuc enzymatic activity and a differential impact on protein
conformation. Overall, through simultaneous monitoring of the impact
of molecular crowding on enzymatic activity and protein conformation,
we reveal a differential impact of molecular crowding on multidomain
proteins containing different linkers and thus aid in further understanding
the impact of molecular crowding on multidomain protein structure
and function.