The macromolecules of the bacterial cell occupy 20-40% of the total cytosol volume, and crowded environments have long been known to compact and stabilize DNA. Nevertheless, investigations on DNA-protein binding are generally performed in the absence of crowding, which may yield an incomplete understanding of how nucleoid-assembling proteins work. A family of such proteins, abundant in Gram-negative bacteria, is the histone-like nucleoid structuring proteins (H-NS). Herein, the synergistic role of macromolecular crowding (mimicked using polyethylene glycol, PEG) and H-NS was investigated using fluorescence correlation spectroscopy (FCS) and enzyme protection assays. We show that crowding enhances the binding of H-NS to the AT-rich tracks of the DNA, where it preferentially binds to, protecting these tracks towards enzyme digestion, inducing some DNA condensation, and inhibiting the biological function of DNA. We further suggest that the looping of DNA chains, induced by H-NS, contributes to the synergistic effect of DNA-binding protein and crowding agents, on DNA condensation.

Four cationic chiral amino acid-based surfactants, cis-and trans-1 and cis-and trans-2, have been studied as DNAcondensing agents with enhanced properties and the absence of cell toxicity. The polar head of the surfactant is made of a cyclobutane β-amino acid in which the amino group is a hydrochloride salt and the carboxyl group is involved in an amide bond, allowing the link with hydrophobic C 12 (surfactant 1) or C 16 (surfactant 2) chains. The ability of these surfactants to condense DNA was investigated using a dye exclusion assay, gel electrophoresis, and circular dichroism and compared with the well-studied dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The surfactant with the longest chain length and the trans stereochemistry (trans-2) was found to be the most efficient in condensing the DNA, including CTAB. Surfactant cis-2 was found to be less efficient, probably due to its poorer solubility. The βamino acid surfactants with the shorter chain length behaved similarly, such that the cis/trans stereochemistry does not seem to play a role in this case. Interestingly, these were also found to induce DNA condensation for the same concentration as trans-2 and CTAB but showed a lower binding cooperativity. Therefore, a longer alkyl chain only slightly improved the effectiveness of these surfactants. Further, atomic force microscopy revealed that they compact DNA into small complexes of about 55−110 nm in diameter.

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in RhoD190N/WT mouse rods with a largely “functionally silenced” rhodopsin mutant to isolate electrical responses triggered by D190N-Rho activity, and found that D190N-Rho at the single-molecule level indeed isomerizes more frequently than WT-Rho by over an order of magnitude. Importantly, however, this higher molecular dark activity does not translate into an overall higher cellular dark noise, owing to diminished D190N-Rho content in the rod outer segment. Separately, we found that much of the degeneration and shortened outer-segment length of RhoD190N/WT mouse rods was not averted by ablating rod transducin in phototransduction—also consistent with D190N-Rho’s higher isomerization activity not being the primary cause of disease. Instead, the low pigment content, shortened outer-segment length, and a moderate unfolded protein response implicate protein misfolding as the major pathogenic problem. Finally, D190N-Rho also provided some insight into the mechanism of spontaneous pigment excitation.

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