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Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1., (© 2024. The Author(s).)

We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' delta1 protons near 100 ppm, and beta and alpha protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the beta protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83-87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of 82 and epsilon1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (approximately 4,000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al. (1988) Inorg. Chem., 37, 4814-4821 ) suggesting that Fe(II)SOD's equatorial His2Asp- ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.

The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5 A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins.

Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487-6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca(2+). The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.