Review
Chemistry, Multidisciplinary
Austin Gamble Jarvi, Xiaowei Bogetti, Kevin Singewald, Shreya Ghosh, Sunil Saxena
Summary: This Account showcases the use of site-directed Cu2+ labeling in proteins and DNA for measuring biomolecular structure and dynamics with electron paramagnetic resonance (EPR) spectroscopy. The labeling scheme provides a straightforward method for obtaining biophysical information that is not accessible using traditional EPR labels. The integration of molecular dynamics (MD) simulations with EPR distance information is expected to advance our understanding of protein and DNA conformational changes and interactions.
ACCOUNTS OF CHEMICAL RESEARCH
(2021)
Article
Chemistry, Physical
Zikri Hasanbasri, Kevin Singewald, Teresa D. Gluth, Benoit Driesschaert, Sunil Saxena
Summary: Sensitive in-cell distance measurements in proteins can be achieved using deuterated OX063 with a maleimide linker (mOX063-d(24)), enabling high protein labeling and more sensitive measurements at 150K compared to 80K. The signal intensity of mOX063-d(24) remains appreciable even in the crowded environment of cells.
JOURNAL OF PHYSICAL CHEMISTRY B
(2021)
Article
Chemistry, Multidisciplinary
Joshua Casto, Alysia Mandato, Lukas Hofmann, Idan Yakobov, Shreya Ghosh, Sharon Ruthstein, Sunil Saxena
Summary: Understanding the structural and mechanistic details of protein-DNA interactions can lead to new ways of combating the virulence of pathogenic bacteria. The study on the Copper Efflux Regulator (CueR) protein reveals that it can bend DNA at high protein concentrations, and this bending enables the coordination with RNA polymerase.
Article
Multidisciplinary Sciences
Vasyl Bondarenko, Marta M. Wells, Qiang Chen, Tommy S. Tillman, Kevin Singewald, Matthew J. Lawless, Joel Caporoso, Nicole Brandon, Jonathan A. Coleman, Sunil Saxena, Erik Lindahl, Yan Xu, Pei Tang
Summary: In this study, using a combination of experiments and computational methods, the full-length intracellular domain (ICD) structure of the human alpha 7 nicotinic acetylcholine receptor in a resting state was determined. It was found that a significant portion of the ICD residues are located in highly flexible regions, with a large loop (loop L) contributing to its stability by anchoring onto the MA helix. Known motifs for cytoplasmic binding, regulation, and signaling were observed in both helices and disordered flexible regions, highlighting the important role of ICD conformational plasticity in orchestrating various biological processes.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Idan Yakobov, Alysia Mandato, Lukas Hofmann, Kevin Singewald, Yulia Shenberger, Lada Gevorkyan-Airapetov, Sunil Saxena, Sharon Ruthstein
Summary: In this study, the dynamical changes of Escherichia coli CueR as a function of copper and DNA binding are measured using EPR spectroscopy. The results show that copper controls the activation of the transcription processes by initiating a series of dynamical changes over the protein.
Article
Biochemistry & Molecular Biology
Kevin Singewald, James A. Wilkinson, Zikri Hasanbasri, Sunil Saxena
Summary: The dynamics of site-specific proteins play a crucial role in protein function. This study utilized EPR spectroscopy to measure the dynamics of specific sites on a protein surface, and validated the results using molecular dynamics simulations. Additionally, small variations were observed at different sites, potentially due to local geometry and electrostatics. This work expands the utility of Cu(II)NTA-based EPR measurements.
Article
Chemistry, Multidisciplinary
Manasseh Kusi Osei, Saber Mirzaei, Xiaowei Bogetti, Edison Castro, Mohammad Azizur Rahman, Sunil Saxena, Raul Hernandez Sanchez
Summary: Template-assisted synthesis of [M-4] clusters was achieved using a tetraamine scaffold L-R(NH2)(4) to direct the formation of C-4-symmetric L-R(NH)(4)Cu-4 clusters. The study showed the importance of metal-metal direct interactions in cluster formation. Computational and experimental analysis indicated the presence of a delocalized electronic structure in the clusters.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2022)
Review
Biophysics
L. Hofmann, A. Mandato, S. Saxena, S. Ruthstein
Summary: Electron paramagnetic resonance (EPR) spectroscopy is a promising structural biology tool to unravel complex and dynamic biological mechanisms. In this article, the advantages of continuous wave (CW) and pulsed EPR distance measurements in resolving transcription processes and protein-DNA interaction are discussed.
BIOPHYSICAL REVIEWS
(2022)
Article
Biochemical Research Methods
Joshua Casto, Xiaowei Bogetti, Hannah R. Hunter, Zikri Hasanbasri, Sunil Saxena
Summary: The narrow excitation bandwidth of monochromic pulses limits the sensitivity of pulsed dipolar spectroscopy on Cu(II)-based measurements. To overcome this limitation, frequency-swept pulses with large excitation bandwidths have been used. In this study, we demonstrate the effectiveness of chirp pulses on commercial instrumentation for Cu(II) distance measurements, and discuss the sensitivity considerations for robust distance measurements using Cu(II) labels in proteins. We show that a sweeping bandwidth chirp pulse can significantly improve the sensitivity of long-range distance measurements, while the sensitivity of short-range distances only slightly increases due to specific considerations for the chirp pulse duration.
JOURNAL OF MAGNETIC RESONANCE
(2023)
Article
Chemistry, Physical
Alysia Mandato, Zikri Hasanbasri, Sunil Saxena
Summary: There have been significant improvements in pulsed ESR sensitivity, making it more applicable for the measurement of biological distance constraints at physiological concentrations and in complex systems. This work demonstrates the power of double quantum coherence (DQC) experiments at Q-band frequencies, enabling rapid and reliable measurements of spin distances at nanomolar concentrations. These nanomolar concentration measurements can lead to further advancements in the use of ESR, especially in cellular contexts.
JOURNAL OF PHYSICAL CHEMISTRY LETTERS
(2023)
Article
Biochemistry & Molecular Biology
Xiaowei Bogetti, Anthony Bogetti, Joshua Casto, Gordon Rule, Lillian Chong, Sunil Saxena
Summary: The catalytic activity of hGSTA1-1 depends on the conformational dynamics of its C-terminal helix a9. The structure of the ligand-free state of the hGSTA1-1 homodimer was determined using EPR distance measurements and WE simulations. Negative cooperativity between the monomers of hGSTA1-1 was observed in the simulations, which suggests its importance in interacting with various toxic substrates.
Review
Chemistry, Multidisciplinary
Xiaowei Bogetti, Sunil Saxena
Summary: This Review discusses computational modeling techniques that enhance the interpretation of Electron Paramagnetic Resonance (EPR) measurements of biomolecules. Molecular dynamics simulations and enhanced sampling strategies are utilized to predict EPR spectra and sample stable conformations of biomolecules and their spin labels. The integration of de novo predictions, EPR measurements, and MD simulations is proposed for efficient sampling of alternate protein conformations. The importance of coupling EPR with modeling tools to investigate protein dynamics and structure is highlighted.
Article
Chemistry, Physical
Zikri Hasanbasri, Nicholas A. Moriglioni, Sunil Saxena
Summary: Combining rigid Cu(ii) labels and pulsed-EPR techniques allows for precise investigation of protein structure and dynamics through distance constraint measurements. The unknown relative orientation of spins in a bilabeled protein poses a challenge due to the orientational selectivity effect. This study dissects the orientational selectivity phenomenon using in silico samples to optimize acquisition schemes and demonstrates the efficiency of rectangular pulses in double electron-electron resonance experiments.
PHYSICAL CHEMISTRY CHEMICAL PHYSICS
(2023)
Review
Chemistry, Analytical
Kevin Singewald, Hannah Hunter, Timothy F. Cunningham, Sharon Ruthstein, Sunil Saxena
Summary: This review focuses on the use of Electron Paramagnetic Resonance (EPR) to measure residue-specific dynamics in proteins, with a specific emphasis on Cu(II)-based spin labels. It discusses the limitations of nitroxide-based spin labels and showcases the use of new Cu(II)-based protein labels, which improve rigidity and expand the spin-labeling toolkit. The review also describes how EPR measurements of the Cu(II) label reflect protein backbone motion and fluctuations, and provides insights into the measurement of site-specific dynamics at both alpha-helices and beta-sheets using the dHis motif. The review concludes by outlining the limitations of the dHis method and suggesting future developments.
ANALYSIS & SENSING
(2023)
Article
Chemistry, Physical
Xiaowei Bogetti, Zikri Hasanbasri, Hannah R. Hunter, Sunil Saxena
Summary: Recent advances in site-directed Cu2+ labeling of proteins and nucleic acids have provided a new methodology for measuring the structure-function relationship in biomolecules. However, accessing the higher sensitivity of Q-band DEER has been challenging for Cu2+ labels designed for proteins. In this study, the orientational effects of the label are analyzed through simulations, and it is shown that three strategically selected magnetic field DEER measurements are generally sufficient for obtaining an orientational-averaged DEER time trace at Q-band.
PHYSICAL CHEMISTRY CHEMICAL PHYSICS
(2022)