Abstract
All biochemical and biophysical processes within a cell occur in a crowded environment. The combined interactions between proteins, DNA, and other large biological macromolecules lead to a complex molecular motion of each object. The complicated nature of all these molecular interactions is often ignored and not investigated in a systematic way. HU protein is one of the most interesting candidates to study the interactions between protein and DNA since it is one of the most abundant proteins in most eubacteria and as a nucleoid-associated protein considered an important factor in shaping the bacterial genome. Through tethered particle motion (TPM) and acoustic force spectroscopy (AFS), optical tweezers combined with a fluorescence confocal microscope, I am able to describe HU-DNA complex in a crowded environment, visualize individual proteins to interact with DNA, and describe the dynamics under different ionic conditions. This is relevant as the DNA binding properties of HU depend on the type of crowding macromolecules and ionic conditions.
Among the above-mentioned techniques, AFS allows force measurements on hundreds of single molecules at the same time. This method is integrated into a lab-on-a-chip device, and allows massively parallel data collection (tens to hundreds of samples), hence yielding well-quantified results. In Chapter 2, I introduce the AFS methodology, including the sample preparation procedure, and demonstrate some of its application of protein-DNA interaction studies.
Most of the studies on protein-DNA interactions have been carried out in a simple in vitro system. It is not evident that these observations can be extrapolated to a living cell. Therefore, I investigated the DNA binding properties under conditions approximating physiological conditions, including macromolecular crowding and salt conditions. Mg2+ plays an important role in the catalytic activity of enzymes and in change the conformation of some proteins. I use HU as a model system since it is one of the most abundant bacterial DNA binding proteins. Using TPM, I recorded the movements of bead-tethered DNA and protein-DNA complexes in three types of crowding agents, blotting-grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000), as well as in conditions with MgCl2. The results in Chapter 3 show two modes co-exist in a transition phase between these two modes. The structure of the crowding macromolecule is changed due to Mg2+, and consequently yields a different crowding impact compared to in the absence of Mg2+. All these observations are useful references for further studies on proteins in a mimicked in vivo environment.
HU has been indirectly observed to diffuse along with DNA in vivo. However, the number of observations and knowledge about the dynamics of the bound HU on DNA is limited. We have learned from TPM results that the introduction of MgCl2 alters the DNA binding behavior of HU. That is why, in Chapter 4, I used optical tweezers and confocal fluorescence microscopy to visualize the movement of single HU dimers directly on double-stranded DNA in the presence and in the absence of Mg2+.
To resolve the effects of varying charges and tensions on HU, I have obtained parameters to describe the protein movement using a diffusion rotation-coupled equation. The parameters indicate that Mg2+ reduces the free-energy barrier for diffusion or increases the radius of gyration (Rg) of HU protein. HU has a higher affinity to stretched DNA, which enhances the stability during DNA melting. Further investigations aimed at studying specific correlations between structural changes of DNA (e.g. whether it is twisted, straight, bent, or coiled) and HU movement (e.g. sliding, pausing, or hopping) are essential to establish good models describing the complexity of nucleoids and cells.
Original language | English |
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Qualification | PhD |
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Award date | 1 Mar 2021 |
Place of Publication | Amsterdam |
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Print ISBNs | 9789464191219 |
Publication status | Published - 1 Mar 2021 |