Search

The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed Cα/C' connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized 13C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems. The different relaxation properties of Cα and C' nuclei are exploited in CACO vs COCA experiments and the complementarity of the two experiments is used to obtain structural information. The two [Fe2S2]+ clusters containing NEET protein CISD3 and the one [Fe4S4]2+ cluster containing HiPIP protein PioC have been taken as model systems. We show that tailored experiments contribute to decrease the blind sphere around the cluster, to extend resonance assignment of cluster bound cysteine residues and to retrieve details on the topology of the iron-bound ligand residues. [ABSTRACT FROM AUTHOR]

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin–cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron–sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes. [ABSTRACT FROM AUTHOR]

In paramagnetic metalloproteins, longitudinal relaxation rates of 13C′ and 13Cα nuclei can be measured using 13C detected experiments and converted into electron spin-nuclear spin distance restraints, also known as Paramagnetic Relaxation Enhancement (PRE) restraints. 13C are less sensitive to paramagnetism than 1H nuclei, therefore, 13C based PREs constitute an additional, non-redundant, structural information. We will discuss the complementarity of 13C PRE restraints with 1H PRE restraints in the case of the High Potential Iron Sulfur Protein (HiPIP) PioC, for which the NMR structure of PioC has been already solved by a combination of classical and paramagnetism-based restraints. We will show here that 13C R1 values can be measured also at very short distances from the paramagnetic center and that the obtained set of 13C based restraints can be added to 1H PREs and to other classical and paramagnetism based NMR restraints to improve quality and quantity of the NMR information. [ABSTRACT FROM AUTHOR]

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE‐1 is a High Potential Iron‐Sulfur Protein (HiPIP) where the [4Fe‐4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root‐mean‐square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo‐designed metalloproteins by NMR. Databases: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing. [ABSTRACT FROM AUTHOR]