1H, 13C and 15N assignment of the paramagnetic high potential iron\u2013sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1

Inês B Trindade,Ricardo O Louro,Francesca Cantini,Mario Piccioli,Michele Invernici

doi:10.1007/s12104-020-09947-6

Abstract

High potential iron–sulfur proteins (HiPIPs) are a class of small proteins (50–100 aa residues), containing a 4Fe–4S iron–sulfur cluster. The 4Fe–4S cluster shuttles between the oxidation states [Fe4S4]3+/2+, with a positive redox potential in the range (500–50 mV) throughout the different known HiPIPs. Both oxidation states are paramagnetic at room temperature. HiPIPs are electron transfer proteins, isolated from photosynthetic bacteria and usually provide electrons to the photosynthetic reaction-center. PioC, the HIPIP isolated from Rhodopseudomonas palustris TIE-1, is the smallest among all known HiPIPs. Despite their small dimensions, an extensive NMR assignment is only available for two of them, because paramagnetism prevents the straightforward assignment of all resonances. We report here the complete NMR assignment of 1H, 13C and 15N signals for the reduced [Fe4S4]2+ state of the protein. A set of double and triple resonance experiments performed with standardized parameters/datasets provided the assignment of about 72% of the residues. The almost complete resonance assignment (99.5% of backbone and ca. 90% of side chain resonances) was achieved by combining the above information with those obtained using a second set of NMR experiments, in which acquisition and processing parameters, as well as pulse sequences design, were optimized to account for the peculiar features of this paramagnetic protein.

Highlights

High potential iron proteins (HiPIPs), together with ferredoxins are a group of metalloproteins that contain a 4Fe–4S cubane cluster coordinated by four cysteine residues
In bacteria HiPIPs are assembled in the cytoplasm and translocated as holoproteins to the periplasm via
The photoferrotrophic metabolism of R. palustris TIE-1 is mediated by the Pio operon that contains three proteins PioA, PioB and PioC (Jiao and Newman 2007)

Summary

Methods and experiments

PioC was expressed and purified as previously reported (Bird et al 2014). Samples of PioC were produced as unlabeled, single 15N-labeled, double 13C and 15N-labeled samples. The optimization of the 15N-HSQC relies on a number of adjustments (Ciofi-Baffoni et al 2014): the decrease of the INEPT transfer from 2.75 ms to values below 1 ms (typically 600–800 μs); the removal the inverse INEPT transfer delay and the signal acquisition in the antiphase mode; the substitution of water selective pulses during the Watergate with the binomial 3–9–19 1H 180° pulse (Mori et al, 1995); the use of states or states-TPPI instead of the echo–antiecho quadrature detection; the use of pulsed field gradients as short as 200 μs; very short recycle delay in order to suppress water signal via progressive saturation and to collect a very large number of scans within reasonable experimental time; adjust the number of t1 and t2 points according to the relaxation properties of “target” resonances Thanks to these adjustments, the 10 HN resonances that were missing in the standard experiments could. Their analysis shows that only a small α-helix in the N-term region is observed in residues 7–11

Conclusion

Findings

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