Enzyme flexibility is known to be crucial for various enzymatic functions. Conformational rearrangements have wide range of timescales, and are considered intrinsic features of all proteins. X-ray crystallography has provided important insight into different enzymatic conformations, but it can only give a snapshot of a complex protein life cycle. Fluorescence spectroscopy methods are becoming the primary methods in investigating protein structural dynamics, especially when combined with different single-molecule techniques.Restriction endonucleases are important tools for every day laboratory work, being essential for all genetic engineering techniques. They are also model systems for studying protein/DNA interactions and one of the best studied families of proteins/enzymes. The main aim of this study was to investigate conformational changes of restriction enzyme BsoBI, during substrate binding and cleavage. Fluorescence resonance energy transfer (FRET) was used to determine the changes in distances between donor and acceptor fluorophores during substrate binding and cleavage, in steady-state or pre-steady state ensemble, as well as in single-molecule experiments. These fluorophores were attached to single cysteine residues introduced to BsoBI subunits at specific positions.BsoBI is a thermostable restriction enzyme, with optimal catalytic activity at 65 °C. Crystallographic studies had shown that BsoBI exists as a homodimer, which completely encircles specific DNA. The substrate is enclosed in a 20 Å long tunnel formed by the protein, which excludes access of water molecules from the solvent. Up to date, only the co-crystal structure of BsoBI with specific DNA is known, and there are no data indicating the conformational changes required for substrate binding and cleavage. The crystallographic data had shown that the interface between the catalytic domains of BsoBI is much weaker than the one between the helical domains, and it was proposed that the major conformational changes happen in the catalytic domain of the enzyme.Steady-state experiments using double labeled BsoBI confirmed that the binding of DNA causes a conformational change in the catalytic domain of the enzyme. The addition of unlabeled specific DNA to the double labeled BsoBI variant A153C (positioned at the catalytic domain) caused a decrease in FRET signal. This result unexpectedly suggested that the fluorophores at the 153 position move apart upon specific substrate binding. The addition of DNA to the double labeled BsoBI variant E100C (positioned at the helical domain) did not cause change in the FRET signal, suggesting that this part of the enzyme does not change its conformation upon substrate binding. Suggested conformational change in the catalytic domain of BsoBI does not involve expected scissor-like motion of two subunits, but more complex twisting motion. These experiments also showed the influence of Ca2+ and Mg2+ ions on the conformation of the catalytic domain of BsoBI. The presence of Ca2+ induced a conformational change of the catalytic domain of the enzyme that moved two fluorophores at the positions 153 closer together, while the presence or the absence of Mg2+ did not cause any effect on the BsoBI conformation.The conformational changes during substrate binding and cleavage, as well as the influence of different metal ions were analysed in pre-steady state experiments, using double labeled BsoBI and unlabeled DNA, or single labeled BsoBI and DNA. These experiments also confirmed the influence of DNA binding on the conformation of the catalytic domain of BsoBI. In addition, the kinetic model of DNA binding and cleavage revealed the existence of at least two conformations of the free enzyme before DNA binding. These two conformations were proposed to be substrate binding competent and substrate binding incompetent. The kinetic models also suggested that BsoBI binds Ca2+ and Mg2+ with different affinity, namely that Ca2+ binding is stronger and faster than Mg2+ binding.The single-molecule experiments confirmed the existence of at least two different conformations of the apo-enzyme, and allowed quantifying the relative proportions of these subpopulations. The analysis of single-molecule experiments using different time windows demonstrated slow dynamics of different conformations of apo-enzyme, which was expected based on the observed influence of temperature on the KM value of BsoBI. Also, it was shown that the enzyme has more than one conformation when bound to the unspecific long substrate, suggesting different binding modes to the unspecific DNA.By combining different fluorescence techniques, as well as different single cysteine variants, it was possible to explain and model single the conformational dynamics of substrate binding and cleavage by BsoBI. These experiments showed unexpected mechanism of the conformational changes, as well as the influence of different metal ions on the conformation of BsoBI.
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