Improved in-cell structure determination of proteins at near-physiological concentration

Abstract

Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.

Cancer/Testis Antigens: “Smart” Biomarkers for Diagnosis and Prognosis of Prostate and Other Cancers

“…Although earlier on there was some skepticism about the existence of disorder in proteins in the crowded cellular environment, this has been refuted by several studies that demonstrate that IDPs remain disordered in vivo both in bacterial and mammalian cells using in-cell NMR [153,154,155]. Clearly, conducting detailed structural and functional characterization of CTAs in vitro and in vivo represents an important future direction in this field.…”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…91 Simple arguments based on a volume exclusion effect due to crowding focus on a shift in populations toward compact states on an otherwise largely unchanged landscape, 11 but this view conflicts with recent experiments. 17,22,23,73,92,93 Largely unaltered conformations in vivo vs in vitro are seen with in-cell solution NMR 40,44 and solid-state (SS) NMR, 54 but other evidence from experiments and simulations indicates that cellular environments may remodel the folded or unfolded ensemble or both 16,17,49,71,94−100 with potentially varying effects in different cellular compartments. 82 This suggests a more complicated picture where it is unclear whether remodeling of the ensemble is again driven mostly by entropic volume exclusion effects or by more enthalpic effects due to interactions with protein crowders and other components of the biological environment.…”

High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

“…For elucidating the subtle difference between the structures in vitro and in eukaryotic cells, it remains necessary to achieve de novo 3D protein structure determination from NOE‐derived distance restraints between side‐chains. Therefore, we improved the procedure that had been utilized for proteins at approximately a concentration of 250 μ m in E. coli cells . Herein we show the first de novo protein structure determinations with high accuracy and precision in living eukaryotic cells.…”

“…3D structures were first calculated with the program CYANA using NOE information as well as backbone dihedral angle restraints derived from chemical shifts. The resulting structure was further refined with the assistance of Bayesian inference using the CYBAY module in CYANA, which was essential for the accurate structure determinations in sf9 cells. 189 NOE‐derived distance restraints, including 54 long‐range restraints, were used (Supporting Information, Table S1).…”