| 1. |
- Albinsson, Bo, 1963, et al.
(författare)
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Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks
- 2016
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Ingår i: Fluorescent Analogues of Biomolecular Building Blocks: Design and Applications: Design and Applications (eds M. Wilhelmsson and Y. Tor). - Hoboken, NJ, USA : John Wiley & Sons, Inc. - 9781118175866 ; , s. 40-54
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- © 2016 by John Wiley & Sons, Inc. All rights reserved. This chapter considers linear dichroism (LD), magnetic circular dichroism (MCD), and Forster resonance energy transfer (FRET) and how these phenomena are related to and may report on transition moment orientations and on molecular structure. MCD is an asset in combination with LD and fluorescence anisotropy for the deciphering of transition moment directions in biophysically important chromophores, and many examples of applications are found among nucleobases. A property corresponding to the absorption anisotropy (linear dichroism) for emission is the fluorescence anisotropy. The amino acid tryptophan, with indole as its photoactive aromatic chromophore, is the most frequently used natural fluorescent probe in biophysical contexts, due to its relatively high fluorescence quantum yield and well-resolved absorption profile in the near-UV. The single-molecule fluorescence-detected linear dichroism (smFLD) method is a useful complement to smFRET since it can help to avoid misinterpretation of false smFRET signals.
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| 2. |
- Beke-Somfai, Tamas, 1977, et al.
(författare)
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Double-lock ratchet mechanism revealing the role of alpha SER-344 in FoF1 ATP synthase
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:12, s. 4828-4833
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Tidskriftsartikel (refereegranskat)abstract
- In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP + Pi. ATP + H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the gamma P-O-ADP bond-forming step via a double-proton transfer. At this step, the highly conserved alpha S344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar PO3- ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.
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| 3. |
- Beke-Somfai, Tamas, 1977, et al.
(författare)
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Energy phase shift as mechanism for catalysis
- 2012
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Ingår i: Chemical Physics Letters. - : Elsevier BV. - 0009-2614. ; 535, s. 169-172
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Tidskriftsartikel (refereegranskat)abstract
- Catalysts are agents that by binding reactant molecules lower the energy barriers to chemical reaction. After reaction the catalyst is regenerated, its unbinding energy recruited from the environment, which is associated with an inevitable loss of energy. We show that combining several catalytic sites to become energetically and temporally phase-shifted relative to each other provides a possibility to sustain the overall reaction by internal 'energy recycling', bypassing the need for thermal activation, and in principle allowing the system to work adiabatically. Using an analytical model for superimposed, phase-shifted potentials of F-1-ATP synthase provides a description integrating main characteristics of this rotary enzyme complex.
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| 4. |
- Beke-Somfai, Tamas, 1977, et al.
(författare)
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Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 49:3, s. 401-403
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Tidskriftsartikel (refereegranskat)abstract
- Despite exhaustive chemical and crystal structure studies, the mechanistic details of how F o F 1 -ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO 3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in β TP and, thus, promote ATP. © 2009 American Chemical Society.
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| 5. |
- Beke-Somfai, Tamas, 1977, et al.
(författare)
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Rate of hydrolysis in ATP synthase is fine-tuned by alpha-subunit motif controlling active site conformation
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:6, s. 2117-2122
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Tidskriftsartikel (refereegranskat)abstract
- Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. FoF1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F-1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central gamma-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-angstrom-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to.-subunit position, the active site conformation is fine-tuned mainly by small alpha-subunit changes. Quantum mechanics-based results confirm that the sub-Angstrom gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.
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| 6. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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A stretched conformation of DNA with a biological role?
- 2017
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 50, s. UNSP e11-e11
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Tidskriftsartikel (refereegranskat)abstract
- We have discovered a well-defined extended conformation of double-stranded DNA, which we call Sigma-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change Delta G = 1.57 +/- 0.12 kcal (mol base pair)(-1) 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3'-3' strands, undergo a sharp transition to the 1.52 +/- 0.04 times longer Sigma-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Sigma-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Sigma-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.
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| 7. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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Force-induced melting of DNA-evidence for peeling and internal melting from force spectra on short synthetic duplex sequences
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:12, s. 8083-8091
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Tidskriftsartikel (refereegranskat)abstract
- Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.
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| 8. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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Tension induces a base-paired overstretched DNA conformation
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:38, s. 15179-15184
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Tidskriftsartikel (refereegranskat)abstract
- Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271: 792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271: 795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106: 18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.
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| 9. |
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| 10. |
- Börjesson, Karl, 1982, et al.
(författare)
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Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies
- 2010
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 4:9, s. 5037-5046
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Tidskriftsartikel (refereegranskat)abstract
- We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption.
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