Achilleas Kapanidis - Academia.edu (original) (raw)

Papers by Achilleas Kapanidis

Research paper thumbnail of Structural organization of RNA polymerase RNA polymerase-promoter complexes: Fluorescence resonance energy transfer

Research paper thumbnail of Closing and opening of the RNA polymerase trigger loop

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active cen... more The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription e...

Research paper thumbnail of RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

Nucleic Acids Research

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains o... more The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence o...

Research paper thumbnail of Single-Molecule Imaging of Transcription, Chromosome Organization, and DNA Repair in Live Bacteria

Biophysical Journal, 2016

impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel re... more impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel reconstitution technique, we demonstrated that monomeric asyn both reversibly blocks and translocates through the voltage-dependent anion channel (VDAC) at nanomolar concentrations. Considering VDAC's major role in regulating metabolite fluxes across the MOM, a functional interaction between a-syn and VDAC could be essential for physiological adaptation of mitochondria and dysfunction in PD. Here we show that the asyn-VDAC interaction is modulated by membrane lipid composition relevant to MOM. We have found that the on-rate of VDAC blockage by a-syn increases up to 10-fold with increase of phosphoethanolamine (PE) content in phosphocholine (PC) membranes. Remarkably, the off-rate was also lipid-dependent, as seen by a 5 mV increase of the turnover potential separating regimes of blockage and translocation with the increase of PE content. We have also found that at physiologically low salt concentrations, the on-rates increase more than 10-fold compared to experiments performed in high salt, while translocation of a-syn through VDAC is impeded. a-Syn differential binding to lipid membranes was also tested using independent methods. These results suggest that the blockage of VDAC by a-syn involves a-syn interaction with the membrane and is governed by both hydrophobic and electrostatic components. Such evidence provides further support for our hypothesis that a-syn needs to bind to the membrane prior to blocking and translocating through the VDAC pore. We propose a new regulatory role of mitochondrial lipids in the (patho-)physiology of monomeric a-syn interaction with mitochondria.

Research paper thumbnail of Structural organization of RNA polymerase RNA polymerase-promoter complexes: Fluorescence resonance energy transfer

Research paper thumbnail of Closing and opening of the RNA polymerase trigger loop

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active cen... more The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription e...

Research paper thumbnail of RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

Nucleic Acids Research

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains o... more The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence o...

Research paper thumbnail of Single-Molecule Imaging of Transcription, Chromosome Organization, and DNA Repair in Live Bacteria

Biophysical Journal, 2016

impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel re... more impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel reconstitution technique, we demonstrated that monomeric asyn both reversibly blocks and translocates through the voltage-dependent anion channel (VDAC) at nanomolar concentrations. Considering VDAC's major role in regulating metabolite fluxes across the MOM, a functional interaction between a-syn and VDAC could be essential for physiological adaptation of mitochondria and dysfunction in PD. Here we show that the asyn-VDAC interaction is modulated by membrane lipid composition relevant to MOM. We have found that the on-rate of VDAC blockage by a-syn increases up to 10-fold with increase of phosphoethanolamine (PE) content in phosphocholine (PC) membranes. Remarkably, the off-rate was also lipid-dependent, as seen by a 5 mV increase of the turnover potential separating regimes of blockage and translocation with the increase of PE content. We have also found that at physiologically low salt concentrations, the on-rates increase more than 10-fold compared to experiments performed in high salt, while translocation of a-syn through VDAC is impeded. a-Syn differential binding to lipid membranes was also tested using independent methods. These results suggest that the blockage of VDAC by a-syn involves a-syn interaction with the membrane and is governed by both hydrophobic and electrostatic components. Such evidence provides further support for our hypothesis that a-syn needs to bind to the membrane prior to blocking and translocating through the VDAC pore. We propose a new regulatory role of mitochondrial lipids in the (patho-)physiology of monomeric a-syn interaction with mitochondria.