Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List

Full text release has been delayed at the author's request until May 08, 2028

ETD Abstract Container

Abstract Header

Structural Studies of Biomolecules using Dynamic Nuclear Polarization and Paramagnetic Solid-State NMR

Bhai N V, Lakshmi
http://rave.ohiolink.edu/etdc/view?acc_num=osu1672854896740666

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Chemical Physics.
Although Magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (SSNMR) spectroscopy has made remarkable progress in structure determination and dynamics studies of biomacromolecules, detection sensitivity has always been a challenge for the solid-state NMR. Hyperpolarization techniques like Dynamic Nuclear Polarization (DNP) can improve the sensitivity of NMR. In DNP, more significant polarization of electron spins is transferred to nuclear spin in the direct vicinity upon saturation of EPR transitions by microwave irradiation. Nuclear polarization is then spread by homonuclear spin diffusion. These polarization transfers can offer a theoretical maximum of 660- fold enhancements to the NMR signal. Thus, DNP has been successfully used to detect signals from biomolecules undetectable by solid-state NMR, such as DNA samples. We used DNP to probe structural restraints from one such DNA sample in the first project. Base pairing in double-stranded DNA usually exists in the canonical Watson-Crick (WC) conformation. However, various DNA complexes with protein and small molecules adopt an alternate Hoogsteen (HG) conformation. This conformational change occurs due to changes in Hydrogen-bonding between purines and pyrimidines. This study compares the Hydrogen-bond strength of WC and HG by accurately measuring the Hydrogen-bonded H-N bond length. While detecting Hoogsteen base pairs is challenging for traditional structural biology techniques, previous studies from our lab have observed Watson-Crick and Hoogsteen base pairs with site-specific resolution with DNP Solid-State NMR. Therefore, in the first project, DNP Solid-State NMR experiments and SIMPSON spectral fitting were used to measure H-N dipolar coupling. The Hydrogen-bonded H-N bond lengths calculated from these dipolar coupling measurements for WC and HG are comparable, indicating that the Hydrogen-bonded N-H bond lengths do not change with the DNA double helix conformation change. Hence, the strength of the Hydrogen bond is conserved with confirmation change. Paramagnetic solid-state NMR has considerably increased the number and length scale of accessible structural restraints compared to conventional dipolar coupling based solid-state NMR. Extending paramagnetic relaxation enhancement (PRE) methodologies to DNP enhanced solid-state NMR could facilitate probing long-range structural restraints in biomacromolecules with improved sensitivity. Also, the ultra-low temperature of the DNP experiments could facilitate PRE measurements of regions of proteins with higher relaxation, like the side chain. The effect of Temperature and DNP conditions on PRE the electron longitudinal relaxation time constant, T1e, for paramagnetic centers Cu2+ and Ni2+ are quantified by using 1H,13C,15N-Labeled Phenylalanine GB1selectively 1H,13C,15N-labeled Phenylalanine GB1 protein. The paramagnetic centers and the diamagnetic analog Zn2+ were incorporated into the protein using N-[S-(2-pyridylthio) cysteaminyl]EDTA tag. The effect of polarizing agent in PRE was studied by varying the concentration of AMUPol. An 8mM AMUPol concentration was used as a comprise between DNP efficiency and additional PRE due to polarizing agent. The T1e is calculated from the ratio of amide 15N longitudinal PRE and 13Cα transverse PRE and is equal to 34ns compared to 2.5ns at room temperature for Cu2+, and 13ns compared to 1ps at room temperature for Ni2+. We proved that using an optimized polarizing agent concentration PRE measurements can be used as a viable method for distance measurements in DNP solid-state NMR. The third study advances paramagnetic solid-state NMR methodologies by probing structural restraints from very large biomolecule TET2, a half-megadalton large dodecameric aminopeptidase. Recent studies have determined the atomic-resolution structure of TET2 by integrating NMR and cryo-EM, and the thermal stability allows TET2 to be expressed and purified with a high yield. Therefore, along with the absence of an intrinsic cysteine, TET2 is an excellent standard to study paramagnetic methodologies in the presence of covalently bound paramagnetic tags which attach site-specifically to cysteine. We determined the ideal tag position for the initial studies by considering solvent accessibility and intra-monomer and inter-monomer distances from the paramagnetic center. Initial PRE studies were conducted using MTSL nitroxide tag and Cu2+ bound to N-[S-(2-pyridylthio) cysteaminyl]EDTA as the paramagnetic centers. The tag attachment in each case was confirmed using 13C-15N correlation spectra acquired at 15kHz MAS in 800MHz spectrometer. However, in each case, the measured PREs were much higher than the expected values, indicating inter-molecular PREs from the paramagnetic centers. Further studies need to be done by diluting the protein with paramagnetic centers with natural abundant wild type protein TET2 to avoid these inter-molecular PREs to probe accurate structural information from the TET2 protein.
Christopher Jaroniec (Advisor)
Bern Kohler (Committee Member)
Rafael Bruschweiler (Committee Member)
178 p.
Biochemistry; Chemistry; Molecular Biology
DNP, NMR spectroscopy, Protein structure, DNA, Biomolecules

Recommended Citations

Citations

  • Bhai N V, L. (2023). Structural Studies of Biomolecules using Dynamic Nuclear Polarization and Paramagnetic Solid-State NMR [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1672854896740666

    APA Style (7th edition)

  • Bhai N V, Lakshmi. Structural Studies of Biomolecules using Dynamic Nuclear Polarization and Paramagnetic Solid-State NMR. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1672854896740666.

    MLA Style (8th edition)

  • Bhai N V, Lakshmi. "Structural Studies of Biomolecules using Dynamic Nuclear Polarization and Paramagnetic Solid-State NMR." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu1672854896740666

    Chicago Manual of Style (17th edition)

osu1672854896740666
© 2023, some rights reserved.Creative Commons License Structural Studies of Biomolecules using Dynamic Nuclear Polarization and Paramagnetic Solid-State NMR by Lakshmi Bhai N V is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.
Release 3.2.12