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

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.publishersversionpublishe

Measuring transverse relaxation in highly paramagnetic systems

The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50–400 s−1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues.publishersversionpublishe

A non-systematic approach

Funding Information: This work benefited from access to CERM/CIRMMP, the Instruct-ERIC Italy centre. Financial support was provided by European EC Horizon 2020 TIMB3 (Project 810856) Instruct-ERIC (PID 4509). This article is based upon work from COST Action CA15133, supported by COST (European Cooperation in Science and Technology) . Fondazione Ente Cassa di Risparmio di Firenze ( CRF 2016 0985 ) is acknowledged for providing fellowship to MI. This work was funded by national funds through FCT– Fundação para a Ciência e a Tecnologia , I.P., Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and Fundação para a Ciência e a Tecnologia (FCT) Portugal is acknowledged for Grant PD/BD/135187/2017 to IBT. Funding Information: This work benefited from access to CERM/CIRMMP, the Instruct-ERIC Italy centre. Financial support was provided by European EC Horizon 2020 TIMB3 (Project 810856) Instruct-ERIC (PID 4509). This article is based upon work from COST Action CA15133, supported by COST (European Cooperation in Science and Technology). Fondazione Ente Cassa di Risparmio di Firenze (CRF 2016 0985) is acknowledged for providing fellowship to MI. This work was funded by national funds through FCT? Funda??o para a Ci?ncia e a Tecnologia, I.P. Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and Funda??o para a Ci?ncia e a Tecnologia (FCT) Portugal is acknowledged for Grant PD/BD/135187/2017 to IBT. Publisher Copyright: © 2020 The Author(s) Copyright: Copyright 2020 Elsevier B.V., All rights reserved.The complete assignment of 1H, 13C and 15N protein signals, which is a straightforward task for diamagnetic proteins provided they are folded, soluble and with a molecular mass below 30,000 Da, often becomes an intractable problem in the presence of a paramagnetic center. Indeed, the hyperfine interaction quenches signal intensity; this prevents the detection of scalar and dipolar connectivities and the sequential assignment of protein regions close to the metal ion(s). However, many experiments can be optimized and novel experiments can be designed to circumvent the problem and to revive coherences invisible in standard experiments. The small HiPIP protein PioC provides an interesting case to address this issue: the prosthetic group is a [Fe4S4]2+ cluster that is bound to the 54 amino acids protein via four cysteine residues. The four cluster-bound cysteine residues adopt different binding conformations and therefore each cysteine is affected by paramagnetic relaxation to different extent. A network of tailored experiments succeeded to obtain the complete resonance assignment of cluster bound residues.publishersversionpublishe

13C Derived Paramagnetic Relaxation Enhancements Are an Additional Source of Structural Information in Solution

Funding Information: This project was supported from the PRIN2020-FAITH (Post-doctoral Fellowship to JMS). This work was funded by National funds through FCT-Fundaҫão para a Ciência e a Tecnologia, I. P. (FCT), project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and LS4FUTURE Associated Laboratory (LA/P/0087/2020). This work benefited from access to CERM/CIRMMP, the Instruct-ERIC Italy centre and from COST Action CA21115, supported by COST (European Cooperation in Science and Technology). Publisher Copyright: © 2023 by the authors.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.publishersversionpublishe

an alternative approach in highly paramagnetic systems

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).authorsversionpublishe

Anion Binding by Dimetallic Nickel(II) and Nickel(III) Complexes of a Face-to-Face Bicyclam: Looking for a Bimacrocyclic Effect

The dinickel­(II) complex of the face-to-face bicyclam ligand α,α′-bis­(5,7-dimethyl-1,4,8,11-tetraazacyclotetradecan-6-yl)-<i>o</i>-xylene (L∩L) in a dimethyl sulfoxide solution exists as a mixture of high- and low-spin forms and uptakes up to three halide and pseudohalide ions (X^–), according to stepwise equilibria, whose constants were determined through spectrophotometric titration experiments. In the case of halides, the first anion goes into the intermetallic cavity, whereas pseudohalides first coordinate the metal center from outside. Comparison with equilibrium data for the complex with the macrocycle 5,7-dimethyl-6-benzyl-1,4,8,11-tetraazacyclotetradecane (L) shows that the dinuclear complex [Ni^II₂(L∩L)]⁴⁺ displays an affinity for the first halide distinctly higher than the mononuclear complex [Ni^II(L)]²⁺, thus disclosing the existence of a <i>bimacrocyclic effect</i> for anion binding. Differential pulse voltammetry studies typically showed a three-peak profile: the most anodic pertaining to the [Ni^II₂(L∩L)]⁴⁺ → Ni^III₂(L∩L)]⁶⁺ two-electron process, then one originating from the [Ni^II₂(L∩L)­X]³⁺ → Ni^III₂(L∩L)­X]⁵⁺ two-electron process, and one deriving from the two two-electron half reactions [Ni^II₂(L∩L)­X₂]²⁺ → Ni^III₂(L∩L)­X₂]⁴⁺ and [Ni^II₂(L∩L)­X₃]⁺ → Ni^III₂(L∩L)­X₃]³⁺, taking place at nearly the same potential. The crystal structure of the [Ni^II₂(L∩L)­(μ-NCO)­(NCO)₂]­ClO₄·2.5H₂O complex salt showed a caterpillar arrangement of the three metal-bound cyanate ions