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

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

Two-dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa

AbstractThe full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551

A portable X-ray fluorescence device for in situ analyses of mural paintings

Within the context of a research program for studying with nondestructive methods the preparation and realisation techniques of mural paintings used in the Roman age, a portable ED-XRF device for elemental analysis has been set up. Preliminary tests have been carried out on two Roman mural paintings belonging to the Baia Archaeological Complex (Naples). Datable to different periods, the two paintings show different manufacturing and conservation states, thus representing a good test for verifying the ED-XRF device capability. In situ and laboratory measurements, performed on materials of different origin and nature, have permitted the determination of the pigments and the recognition of pollution traces

A portable X-ray fluorescence device for in situ analyses of mural paintings

Within the context of a research program for studying with nondestructive methods the preparation and realisation techniques of mural paintings used in the Roman age, a portable ED-XRF device for elemental analysis has been set up. Preliminary tests have been carried out on two Roman mural paintings belonging to the Baia Archaeological Complex (Naples). Datable to different periods, the two paintings show different manufacturing and conservation states, thus representing a good test for verifying the ED-XRF device capability. In situ and laboratory measurements, performed on materials of different origin and nature, have permitted the determination of the pigments and the recognition of pollution traces

Biomolecular NMR at 1.2 GHz

The development of new superconducting ceramic materials, which maintain the superconductivity at very intense magnetic fields, has prompted the development of a new generation of highly homogeneous high field magnets that has trespassed the magnetic field attainable with the previous generation of instruments. But how can biomolecular NMR benefit from this? In this work, we review a few of the notable applications that, we expect, will be blooming thanks to this newly available technology.Comment: 17 pages, 13 figure