How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical−chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.in-cell NMR | protein surface properties | intracellular diffusion D espite considerable progress in mapping out how proteins interact functionally through structure and evolved interfaces (1-3), there is yet little known about how proteins interact nonspecifically upon random diffusive encounters (4-10). Although these nonspecific "quinary" (11) interactions are typically weak and short-lived, they are still expected to affect function because of their sheer numbers: Under crowded cellular conditions, they compete with specific binding (6-8), control diffusion (12), and skew structural stability (5,(13)(14)(15)(16)(17)(18)(19). The question is then to what extent this dynamic background of nonspecific interactions is biologically controlled and optimized. Part of the answer is hinted by the tendency of soluble proteins, nucleic acids, and membranes to carry a repulsive net-negative charge (20,21 , and PO 4 2− (22). However, proteins expose also positive, polar, and hydrophobic moieties that operate against the net-negative charge repulsion by engaging in attractive interactions upon diffusive encounters. The strength and duration of these attractive interactions depend on the proteins' detailed surface composition, relative orientations, and ability to adapt complementary shapes. Following Elcock's estimate for the Escherichia coli cytoplasm, each protein experiences at all times approximately five putative interaction partners in its immediate cellular environment (8). Sometimes, mutual fits enable strong functional binding (1, 2), but, most often, the proteins just separate after a brief tête-à-tête (3), in search of higher-affinity partners. A key detail is that the eff...

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.