Measuring Protein Shapes in Living Cells

Proteins fold into intricate shapes, known as conformations. The activation of many signal transduction proteins, kinases, and transcription factors requires a change in their conformations. Thus the conformation of a protein can indicate its biological activity. This importance of conformational changes has stimulated the development of numerous methods for analyzing protein conformations and interactions, such as native mass spectrometry¹ and cryoelectron microscopy.² These methods may achieve detailed characterizations of protein conformations, but they require highly purified proteins; they are challenged by the complexity of in vivo proteomes.

To enable the in vivo analysis of protein conformations, Bamberger et al.³ report a covalent protein painting (CPP) of lysine residues, which directly determines the lysine residues that are accessible for covalent modification on protein surfaces. Specifically, the accessible amine groups on lysine residues are dimethylated in living cells, whereas the inaccessible amine groups are dimethylated with isotopically distinct methyl groups after protein extraction and digestion. Mass spectrometry analysis can distinguish and accurately quantify the peptides and their dimethyl labels, thus providing direct information for the fraction of lysine residues exposed on protein surfaces.³ Therefore, CPP is a type of protein footprinting⁴ approach that offers advantages over other footprinting methods. For example, CPP allows for the fast and highly specific covalent labeling of an amino acid that is frequently exposed on protein surfaces. A related footprinting method⁵ uses tandem mass tags (TMT) to analyze the lysine surface accessibility, but it cannot be applied to living cells because cell membranes are not permeable to TMT. In contrast, the dimethyl labels used by CPP permeate cell membranes and allow labeling and analyzing the entire proteome of living cells without requiring protein purification. This ability to perform in vivo labeling offers the exciting potential to use CPP to quantify protein conformational changes in tissues of animal models.

The efficiency and specificity of dimethyl labeling can confer high-sensitivity to CPP and perhaps even enable single-cell sensitivity. One approach to this goal might include using isobaric carriers⁶ to increase the sensitivity of CPP toward single-cell analysis. Then, the protein surface accessibility measured by CPP may provide information for protein activities and binding interactions, thus contributing a vitally needed dimension to single-cell analysis.⁷ CPP will certainly find numerous applications, as hinted by the protein conformational changes in Alzheimer’s disease already reported by Bamberger et al.³