Browsing by Subject "Facilitated dissociation"

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Open Access
Facilitated dissociation of nucleoid-associated proteins from DNA in the bacterial confinement
(Biophysical Society, 2022-04-05) Koşar, Zafer; Attar, A. Göktuğ; Erbaş, Aykut
Transcription machinery depends on the temporal formation of protein-DNA complexes. Recent experiments demonstrated that not only the formation but also the lifetime of such complexes can affect the transcriptional machinery. In parallel, in vitro single-molecule studies showed that nucleoid-associated proteins (NAPs) leave the DNA rapidly as the bulk concentration of the protein increases via facilitated dissociation (FD). Nevertheless, whether such a concentration-dependent mechanism is functional in a bacterial cell, in which NAP levels and the 3d chromosomal structure are often coupled, is not clear a priori. Here, by using extensive coarse-grained molecular simulations, we model the unbinding of specific and nonspecific dimeric NAPs from a high-molecular-weight circular DNA molecule in a cylindrical structure mimicking the cellular confinement of a bacterial chromosome. Our simulations confirm that physiologically relevant peak protein levels (tens of micromolar) lead to highly compact chromosomal structures. This compaction results in rapid off rates (shorter DNA residence times) for specifically DNA-binding NAPs, such as the factor for inversion stimulation, which mostly dissociate via a segmental jump mechanism. Contrarily, for nonspecific NAPs, which are more prone to leave their binding sites via 1d sliding, the off rates decrease as the protein levels increase. The simulations with restrained chromosome models reveal that chromosome compaction is in favor of faster dissociation but only for specific proteins, and nonspecific proteins are not affected by the chromosome compaction. Overall, our results suggest that the cellular concentration level of a structural DNA-binding protein can be highly intermingled with its DNA residence time.
Open Access
Protein-DNA dissociation kinetics and chromosome organization in a model bacterial confinement
(2021-09) Koşar, Zafer
Transcriptional initiation and repression require the temporal interactions of transcription factors with DNA. Recent experiments showed that the interaction lifetime is crucial for transcriptional regulation. Relevantly, in vitro single-molecule studies showed that nucleoid-associated proteins (NAPs) dissociate rapidly from DNA through facilitated dissociation (FD) with the increasing phase-solution protein concentration. Nevertheless, it is not clear whether such a concentration-dependent mechanism is functional in bacterial confinement, in which NAP levels and the 3D chromosomal architecture are coupled. Here, we employ extensive coarse-grained molecular simulations, where we model the dissociation of specific and nonspecific dimeric NAPs from a high-molecular-weight circular DNA polymer in a rod-shaped structure constituting the cellular boundaries. Our simulations indicate that the peak cellular protein concentrations result in highly compact chromosomal conformations. Such compactions lead to shorter DNA-residence times but only for NAPs demonstrating sequence-specificity, such as the factor for inversion stimulation (Fis). On the other hand, the dissociation rates of nonspecific NAPs decrease with the increasing protein concentrations, exhibiting an inverse FD behavior. Another set of simulations utilizing restrained chromosome models reveal DNA-segmental fluctuations as the cause of this reversed response, suggesting that moderate chromosomal compaction promotes protein dissociation. Together, our findings suggest that cellular quantities of structural DNA-binding proteins could be highly influential on their residence times and the chromosome architecture.
Open Access
The role of ligand rebinding and facilitated dissociation on the characterization of dissociation rates by surface plasmon resonance (SPR) and benchmarking performance metrics
(Springer, 2022) Erbaş, Aykut; İnci, Fatih; Vanhaelen, Quentin
Surface plasmon resonance (SPR) is a real-time kinetic measurement principle that can probe the kinetic interactions between ligands and their binding sites, and lies at the backbone of pharmaceutical, biosensing, and biomolecular research. The extraction of dissociation rates from SPR-response signals often relies on several commonly adopted assumptions, one of which is the exponential decay of the dissociation part of the response signal. However, certain conditions, such as high density of binding sites or high concentration fluctuations near the surface as compared to the bulk, can lead to non-exponential decays via ligand rebinding or facilitated dissociation. Consequently, fitting the data with an exponential function can underestimate or overestimate the measured dissociation rates. Here, we describe a set of alternative fit functions that can take such effects into consideration along with plasmonic sensor design principles with key performance metrics, thereby suggesting methods for error-free high-precision extraction of the dissociation rates.