Abstract
In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins.
Highlights
Summary, the different patterns of 1H-15N NMR signal recovery of the PFN1 mutants obtained in human and bacterial cells indicate that, while non-specific electrostatic interactions are abundant in both environments, functional interactions contribute to the 1H-15N NMR signal loss in human cells due to the formation of complexes with soluble partners which are absent in bacteria
Recent applications of in-cell NMR have shown that soluble cytoplasmic proteins are often engaged in many weak, non-specific interactions within the cell, which in some cases lead to the complete loss of NMR signals, preventing further analysis
We showed that the contribution of these different kinds of interactions can be investigated by introducing mutations on the appropriate surface residues, and by comparing the in-cell NMR behaviour of each mutant in different cellular environments: Figure 4
Summary
Summary, the different patterns of 1H-15N NMR signal recovery of the PFN1 mutants obtained in human and bacterial cells indicate that, while non-specific electrostatic interactions are abundant in both environments, functional interactions contribute to the 1H-15N NMR signal loss in human cells due to the formation of complexes with soluble partners which are absent in bacteria. Diagrams summarizing the different behaviour of the PFN1 mutants in human (a) and bacterial cells (b), as observed by in-cell 1H-15N SOFAST-HMQC.
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