Quantifying Protein Electrostatic Interactions in Cells by Nuclear Magnetic Resonance Spectroscopy

Abstract

Most proteins perform their functions in cells. How the cellular environment modulates protein interactions is an important question. In this work, electrostatic interactions between protein charges were studied using in-cell nuclear magnetic resonance (NMR) spectroscopy. A total of eight charge pairs were introduced in protein GB3. Compared to the charge pair electrostatic interactions in a buffer, five charge pairs in cells displayed no apparent changes whereas three pairs had the interactions weakened by more than 70%. Further investigation suggests that the transfer free energy is responsible for the electrostatic interaction modulation. Both the transfer free energy of the folded state and that of the unfolded state can contribute to the cellular environmental effect on protein electrostatics, although the latter is generally larger (more negative) than the former. Our work highlights the importance of direct in-cell studies of protein interactions and thus protein function.

Intracellular environment can change protein conformational dynamics in cells through weak interactions

“…S2), suggesting that the cell sample was stable during the relaxation measurements and signals were from GB3L in cells. The cells were viable after NMR experiments as proved previously (67).…”

Bioinspired Ice-Binding Materials for Tissue and Organ Cryopreservation

“…The last decades have witnessed the fast development of high-resolution and in vivo real-time detection techniques in crowded biological environments. For example, researchers have been able to determine the high-resolution three-dimensional structure of proteins inside crowded biological environments for the first time by combining “in-cell” nuclear magnetic resonance spectroscopy and the cutting-edge computational algorithms. , Imaging techniques such as the ambient mass spectrometric imaging, single-molecule localization microscopy, , and chemical imaging technique are becoming ever-more powerful tools for quantitatively visualizing macromolecules in the complex in vivo environment. Fluorescence correlation and cross-correlation spectroscopies have seen a greater rise in the last decades revealing information about flow velocity, protein–protein binding, ligand–receptor affinity, and transport in living organisms. − Linking IBPs with fluorescent molecules, the dynamics of IBPs in their in vivo surroundings can be determined.…”

Aggregates of fluorescent gels assembled by interfacial dynamic bonds

“…Protein phosphorylation adjusts the charge state of side chains after undergoing the posttranslational modification, in this way, the structures of protein could be altered. [ 69 ] When opposite charges are incorporated into fluorophores as supramolecular interaction sites, a variety of AFGs can be constructed based on electrostatic interactions. Meanwhile, due to the sensitivity to pH, temperature, and competitive ions, the resulting AFGs allow themselves to be good candidates for applying to biosystems and medical fields.…”

“…[53,54] The involved supramolecular interactions include H-bond, [55,56] metal coordination interactions, [35,57,58] host−guest interactions, [59][60][61][62][63] hydrophobic interactions, [64][65][66] electrostatic interactions. [67][68][69] In addition to that, dynamic covalent bonds are also employed for interfacial adhesion, principally including the acyl hydrazone bond and imine bond. [70,71] To date, the studies concerning dynamic bondbased AFGs flourished, hence, this review summaries a series of AFGs in accordance with different interfacial dynamic bonds, also shedding light on their applications in distinct realms.…”