Abstract
Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.
Highlights
Many processes in biological cells depend on interactions between macromolecules and on the ability of these macromolecules to find each other by translational diffusion
These findings indicate that the positive GFP variants bind to ribosomes, and that this is the major cause for their slow diffusion
We made a number of assumptions: (1) the exchange between free and bound state is much faster than the fluorescence recovery after photo-bleaching (FRAP) measurement; (2) the highest diffusion coefficient of all variants in a given organism reflects the free state of GFP; (3) GFPs bind solely to ribosomes; (4) the total number of binding sites on all ribosomes is higher than the number of GFPs; (5) the decrease of diffusion coefficient with net positive charge has the same origin in all three organisms; and, (6) the ribosome diffusion coefficient is the same in all three organisms
Summary
Many processes in biological cells depend on interactions between macromolecules (proteins and nucleic acids) and on the ability of these macromolecules to find each other by translational diffusion. This is especially important in prokaryotes because of the virtual absence of active mechanisms of cytoplasmic transport. Examples of diffusion-limited processes are binding of tRNA complexes to the ribosome, which leads to limitation in cell growth (1); and the binding of barstar to barnase, which we know to be diffusion limited because the proteins are designed to have an increased association rate by electrostatic interactions (2). Because protein diffusion is influenced by the environment, we need to determine diffusion coefficients in the context of the cell
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