We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.

Deep mutational scanning is a widely used method for multiplex measurement of functional consequences of protein variants. We developed a new deep mutational scanning statistical model that generates error estimates for each measurement, capturing both sampling error and consistency between replicates. We apply our model to one novel and five published datasets comprising 243,732 variants and demonstrate its superiority in removing noisy variants and conducting hypothesis testing. Simulations show our model applies to scans based on cell growth or binding and handles common experimental errors. We implemented our model in Enrich2, software that can empower researchers analyzing deep mutational scanning data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1272-5) contains supplementary material, which is available to authorized users.

We use U2OS cells as in vivo ‘test tubes’ to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by Fast Relaxation Imaging (FReI), which combines a temperature jump with fluorescence microscopy of FRET-labeled proteins. While the stability of the cytoplasmic enzyme PGK increases in cells, the stability of the cell-surface antigen VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding, relative to their respective in aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VlsE is stabilized by 150 mg/ml of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VlsE in U2OS cells: long range interactions competing with crowding, or shape-dependent crowding, which favors more compact states inside the cell over the elongated aqueous buffer native state.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.