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.