Investigation of site-specific dynamic nuclear polarization on biomolecules

• Applicant: Professor Dr.-Ing. Björn Corzilius
• Subject Area: Analytical Chemistry
• Term: from 2012 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 232503709

• Final Report Year: 2023

Final Report Abstract

The theme of the project was site-specific dynamic nuclear polarization (DNP) of biomolecules. Site-specific DNP promises distance information between the paramagnetic polarizing agent (PA) acting as source of large spin polarization and the targeted nucleus by measurement of enhanced magic-angle spinning (MAS) NMR signals, and may induce specificity and thus intrinsic selectivity of the signals in the NMR spectrum. The project has answered the following problems: (1) How do PAs suitable for targeted DNP, especially metal ions, perform at relevant magnetic fields? (2) What is the distance dependence of direct DNP transfer? (3) Can endogenous paramagnetic sites or attached spin-labels be utilized for site-selective DNP? To address these problems, three work packages (WPs) were conceived, each specifically addressing one of the above mentioned aims. In WP1 we investigated the PA concentration dependence of Gd-DOTA at 9.4 T and found that at higher concentrations cross effect (CE) also becomes an active DNP mechanism besides solid effect (SE) which is solely responsible for DNP at low concentrations (< 5–10 mM). We developed a theoretical description of CE with high-spin metal ions in rotating solids and deciphered the distance dependence of the onset of CE for different nuclei (1H, 13C, 15N) using synthesized bis- (Gd-complex)es. These developments significantly increased our fundamental knowledge of metal-ion DNP and allowed this technique to become an important tool, e.g., for the investigation of battery materials. We also tested N@C60 doped into the neat C60 fullerene matrix as a narrowline PA. Unfortunately, 13C enhancement factors in C60 are relatively small, and become insignificant if N@C60 is doped into any other matrix. Nevertheless, the unique molecular system allowed us to draw important conclusions about the mechanism of homonuclear spin-diffusion in the vicinity of the polarizing electron spin under MAS and can serve as a mode purity probe for gyrotrons or other high-frequency microwave sources used for DNP. WP2 has been hampered by several scientific setbacks. While early results using a tailored electron–nuclear spin system were promising, the quantitative analysis of potentially distant-dependent DNP effects was unsuccessful due to limited solubility in DNP-supporting solvent matrices and dissociation of the bound Gd3+. Directly 13C-labeling Gd-DOTA yielded inconclusive results. Further attempts at designing a DNA double-helix based distance ruler set was delayed due to instrumental issues. Since we finally had a breakthrough in deciphering the electron–nuclear distance dependence in Gd-labeled ubiquitin during the parallel progress of WP3, we did not pursue WP2 any further but instead put our focus on the next WP. However, we have recently combined the knowledge gained in all three WPs into the subproject A2 of TRR 386 which starts in October 2023 and aims at a resolution of the underlying question of WP2. WP3 has proven to provide the most diverse and rewarding research results during the course of the Emmy Noether project. Early approaches towards site-specific DNP within P. aeruginosa LecA where Ca2+ was substituted by Gd3+ or by Gd3+ bound in a lanthanide-binding tag within interleukin-1β were unsuccessful due to compromised metal-ion binding in DNP matrix or low symmetry of the binding site, respectively. The first breakthrough was made by endogenous binding of Mn2+ in a hammerhead ribozyme (HHRz), yielding a direct Mn2+–13C DNP enhancement of 8, emerging only from within the bound complex. Furthermore, site-directed spin labeling of ubiquitin single-site mutants with a Gd3+-chelate label gave us a detailed understanding of the complex competition between direct DNP, spin relaxation and spin diffusion. Through protein deuteration we obtained direct 15N DNP enhancements of > 100, and by combination with computationally assisted conformer analysis we could correlate the specific DNP buildup rate with the average distances of the amino acid side chains to Gd3+, supporting the developed theory and showing that direct 15N DNP over distances of at least 2.7 nm is possible, while nuclei within ~1.2 nm around Gd3+ are NMR-invisible due to paramagnetic resonance quenching. Due to a serendipitous observation made during the investigation of IL1β (which finally proved unsuitable for site-specific DNP, see above), we discovered and theoretically explained the occurrence of an alternative DNP transfer path active during direct DNP with conventional bis-nitroxide PAs. This path relies on active molecular motions (e.g., methyl reorientation) supporting heteronuclear cross relaxation and therefore features an outstanding opportunity to foster sitespecific DNP without detrimental paramagnetic effects. We have since further developed this method for the selective spectroscopy of interfaces and the method has been adopted by several DNP groups worldwide for the investigation of biomolecular systems or materials.

Publications

• Gd(iii) and Mn(ii) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins. Physical Chemistry Chemical Physics, 18(39), 27205-27218.
  Kaushik, Monu; Bahrenberg, Thorsten; Can, Thach V.; Caporini, Marc A.; Silvers, Robert; Heiliger, Jörg; Smith, Albert A.; Schwalbe, Harald; Griffin, Robert G. & Corzilius, Björn
  
• Heteronuclear Cross-Relaxation under Solid-State Dynamic Nuclear Polarization. Journal of the American Chemical Society, 138(51), 16572-16575.
  Daube, Diane; Aladin, Victoria; Heiliger, Jörg; Wittmann, Johannes J.; Barthelmes, Dominic; Bengs, Christian; Schwalbe, Harald & Corzilius, Björn
  
• Theory of solid effect and cross effect dynamic nuclear polarization with half-integer high-spin metal polarizing agents in rotating solids. Physical Chemistry Chemical Physics, 18(39), 27190-27204.
  Corzilius, Björn
  
• Bis‐Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization. Angewandte Chemie, 129(15), 4359-4363.
  Kaushik, Monu; Qi, Mian; Godt, Adelheid & Corzilius, Björn
  
• Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR. Progress in Nuclear Magnetic Resonance Spectroscopy, 102-103(2017, 11), 120-195.
  Lilly Thankamony, Aany Sofia; Wittmann, Johannes J.; Kaushik, Monu & Corzilius, Björn
  
• Paramagnetic Metal Ions for Dynamic Nuclear Polarization, eMagRes 7, 179– 194 (2018)
  Corzilius, B.
  
• Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP. Angewandte Chemie, 131(15), 4917-4922.
  Aladin, Victoria; Vogel, Marc; Binder, Robert; Burghardt, Irene; Suess, Beatrix & Corzilius, Björn
  
• Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy. Journal of the American Chemical Society, 141(50), 19888-19901.
  Mao, Jiafei; Aladin, Victoria; Jin, Xinsheng; Leeder, Alexander J.; Brown, Lynda J.; Brown, Richard C. D.; He, Xiao; Corzilius, Björn & Glaubitz, Clemens
  
• Site-specific dynamic nuclear polarization in a Gd(iii)-labeled protein. Physical Chemistry Chemical Physics, 22(44), 25455-25466.
  Heiliger, Jörg; Matzel, Tobias; Çetiner, Erhan Can; Schwalbe, Harald; Kuenze, Georg & Corzilius, Björn
  
• Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR. Chemical Reviews, 122(10), 9738-9794.
  Biedenbänder, Thomas; Aladin, Victoria; Saeidpour, Siavash & Corzilius, Björn