| 1. |
- Holm, Anne Ivalu Sander, et al.
(författare)
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Vacuum-ultraviolet circular dichroism spectroscopy of DNA : a valuable tool to elucidate topology and electronic coupling in DNA
- 2010
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 12:33, s. 9581-9596
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Tidskriftsartikel (refereegranskat)abstract
- Circular dichroism (CD) is a powerful technique to obtain information on electronic transitions and has been used extensively for studies on DNA. Most experiments are done in the UV region but new information is often revealed from extending the wavelength region down into the vacuum ultraviolet (VUV) region. Such experiments are most easily carried out with synchrotron radiation (SR) light sources that provide large photon fluxes. Here we provide a summary of the SRCD data taken on different DNA strands with emphasis on results from our own laboratory within the last five years.(1-3) Signal intensities in the VUV are often significantly larger than those in the UV, and the electronic coupling between bases may increase with excitation energy. CD spectroscopy is particularly useful for investigating the extent of electronic coupling within a strand, i.e., the degree of delocalisation of the excited-state electronic wavefunction. The spatial extent of the wavefunction may be limited to just one base or it extends over two or more bases in a stack or between bases on different strands.(4,5) The actual character of the electronically excited state is linked to base composition and sequence as well as DNA folding motif (A-, B-, Z-DNA, triplexes, quadruplexes, etc.). The latter depends on experimental conditions such as solution acidity, temperature, ionic strength, and solvent.
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| 2. |
- Karlsson, Elin, et al.
(författare)
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Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
- 2019
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Ingår i: Biophysical Journal. - : CELL PRESS. - 0006-3495 .- 1542-0086. ; 117:4, s. 729-742
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Tidskriftsartikel (refereegranskat)abstract
- Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
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| 3. |
- Kjaer, Christina, et al.
(författare)
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Circular dichroism, anisotropy and absorption spectroscopy of chlorophyll b in methanol and mixed methanol-water solutions
- 2020
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 22:46, s. 26961-26966
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Tidskriftsartikel (refereegranskat)abstract
- The spectroscopic properties of chlorophyll (Chl) strongly depend on interactions with other Chl molecules, a fact that nature exploits in light harvesting by photosynthetic proteins. In solution, complex Chl aggregates are formed that depend not only on the solvent, but also on the detailed preparation procedure. Here we report synchrotron radiation circular dichroism (SRCD) spectra of Chlb in methanol (MeOH) and MeOH/H2O mixtures; in the latter, water molecules assist in the formation of Chl aggregates as Chlb is too hydrophobic to dissolve in water. The magnitude of the most prominent CD signal increases up to 100-fold over time (2-15 hours) when the water content is increased from 0 to 50% in volume, the signal is non-conservative (almost exclusively negative), and sensitive to sample preparation. Three different types of signature CD spectra (Types A to C) are identified depending on preparation, and the change in CD signal over time and with temperature is further analyzed with anisotropy spectroscopy (ratio of simultaneously recorded CD to absorption) and principal component analysis (PCA). We show that CD is clearly superior to pure absorption spectroscopy in identifying structural changes, and anisotropy spectroscopy further increases the sensitivity towards smaller structural changes. PCA on temperature dependent CD data show that depending on preparation, and thus the type of aggregate as revealed by the CD signature, either one (Type A) or two chiral species (Type B) are identified in the spectra, further evidencing the complex nature of Chlb aggregates. Furthermore, the CD signal decreases linearly with volume when a sample of Chlb in MeOH/H2O (i.e., a sample of Chlb aggregates) is diluted, which implies that the aggregation process is irreversible: once aggregates are formed, they largely do not revert back to monomers. However, anisotropy spectroscopy reveals that there are small changes in the aggregates, not directly noticeable in CD and absorption. The work presented here demonstrates, compared to absorption spectroscopy, a clear advantage of CD and anisotropy spectroscopy in studying the complex evolution of Chl samples with time and temperature.
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