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Mobility is a common feature of biomacromolecules, often fundamental for their function. Thus, in many cases, biomacromolecules cannot be described by a single conformation, but rather by a conformational ensemble. NMR paramagnetic data demonstrated quite informative to monitor this conformational variability, especially when used in conjunction with data from different sources. Due to their long-range nature, paramagnetic data can, for instance, i) clearly demonstrate the occurrence of conformational rearrangements, ii) reveal the presence of minor conformational states, sampled only for a short time, iii) indicate the most representative conformations within the conformational ensemble sampled by the molecule, iv) provide an upper limit to the weight of each conformation., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Protein frameworks are an emerging class of biomaterial with medical and technological applications. Frameworks are studied mainly by X-ray diffraction or scattering techniques. Complementary strategies are required. Here, we report solid-state NMR analyses of a microcrystalline protein-macrocycle framework and the rehydrated freeze-dried protein. This methodology may aid the characterization of low-crystallinity frameworks.

Protein solid-state NMR has evolved dramatically over the last two decades, with the development of new hardware and sample preparation methodologies. This technique is now ripe for complex applications, among which one can count bioconjugation, protein chemistry and functional biomaterials. In this review, we provide our account on this aspect of protein solid-state NMR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Field-cycling NMR relaxometry is a well-established technique that can give information on molecular structure and dynamics of biological systems. It provides the nuclear relaxation rates as a function of the applied magnetic field, starting from fields as low as ~ 10 -4 T up to about 1-3 T. The profiles so collected, called nuclear magnetic relaxation dispersion (NMRD) profiles, can be extended to include the relaxation rates at the largest fields achievable with high resolution NMR spectrometers. By exploiting this wide range of frequencies, the NMRD profiles can provide information on motions occurring on time scales from 10 -6 to 10 -9 s. 1 H NMRD measurements have proved very useful also for the characterization of paramagnetic proteins, because they can help characterise a number of parameters including the number, distance and residence time of water molecules coordinated to the paramagnetic center, the reorientation correlation times and the electron spin relaxation time, and the electronic structure at the metal site., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS. Second, the fraction of resonances in the spectrum of a paramagnetic sample that can be paired with the diamagnetic counterparts is critical for the assignment. With the data from two tag positions a reliable assignment could be obtained for 60% of the methyl groups and for many of the remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.

Solid-state NMR is becoming a powerful tool to detect atomic-level structural features of biomolecules even when they are bound to (or trapped in) solid systems that lack long-range three-dimensional order. We here demonstrate that it is possible to probe protein-ligand interactions from a protein-based perspective also when the protein is entrapped in silica, thus translating into biomolecular solid-state NMR all of the considerations that are usually made to understand the chemical nature of the interaction of a protein with its ligands. This work provides a proof of concept that also immobilized enzymes can be used for protein-based NMR protein-ligand interactions for drug discovery.

The binding of paramagnetic metal ions to proteins produces a number of different effects on the NMR spectra of the system. In particular, when the magnetic susceptibility of the metal ion is anisotropic, pseudocontact shifts (PCSs) arise and can be easily measured. They constitute very useful restraints for the solution structure determination of metal-binding proteins. In this context, there has been great interest in the use of lanthanide(III) ions to induce PCSs in diamagnetic proteins, e.g. through the replacement native calcium(II) ions. By preparing multiple samples in each of which a different ion of the lanthanide series is introduced, it is possible to obtain multiple independent PCS datasets that can be used synergistically to generate protein structure ensembles (typically called bundles). For typical NMR-based determination of protein structure, it is necessary to perform an energetic refinement of such initial bundles to obtain final structures whose geometric quality is suitable for deposition in the PDB. This can be conveniently done by using restrained molecular dynamics simulations (rMD) in explicit solvent. However, there are no available protocols for rMD using multiple PCS datasets as part of the restraints. In this work, we extended the PCS module of the AMBER MD package to handle multiple datasets and tuned a previously developed protocol for NMR structure refinement to achieve consistent convergence with PCS restraints. Test calculations with real experimental data show that this new implementation delivers the expected improvement of protein geometry, resulting in final structures that are of suitable quality for deposition. Furthermore, we observe that also initial structures generated only with traditional restraints can be successfully refined using traditional and PCS restraints simultaneously.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Long-range NMR restraints, such as diamagnetic residual dipolar couplings and paramagnetic data, can be used to determine 3D structures of macromolecules. They are also used to monitor, and potentially to improve, the accuracy of a macromolecular structure in solution by validating or "correcting" a crystal model. Since crystal structures suffer from crystal packing forces they may not be accurate models for the macromolecular structures in solution. However, the presence of real differences should be tested for by simultaneous refinement of the structure using both crystal and solution NMR data. To achieve this, the program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic and paramagnetic NMR data and/or diamagnetic residual dipolar couplings. Inconsistencies between crystal structures and solution NMR data, if any, may be due either to structural rearrangements occurring on passing from the solution to solid state, or to a greater degree of conformational heterogeneity in solution with respect to the crystal. In the case of multidomain proteins, paramagnetic restraints can provide the correct mutual orientations and positions of domains in solution, as well as information on the conformational variability experienced by the macromolecule., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramagnetic data, are becoming increasingly popular for the characterization of conformational heterogeneity of multidomain biomacromolecules and protein complexes. The question addressed here is how much information is contained in these averaged data. We have analyzed and compared the information content of conformationally averaged RDCs caused by steric alignment and of both RDCs and pseudocontact shifts caused by paramagnetic alignment, and found that, despite the substantial differences, they contain a similar amount of information. Furthermore, using several synthetic tests we find that both sets of data are equally good towards recovering the major state(s) in conformational distributions.

Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This "tuned" flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

The presence of heterogeneity in the interdomain arrangement of several biomolecules is required for their function. Here we present a method to obtain crucial clues to distinguish between different kinds of protein conformational distributions based on experimental NMR data. The method explores subregions of the conformational space and provides both upper and lower bounds of probability for the system to be in each subregion.

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.

We have proposed solid state NMR (SSNMR) of sedimented solutes as a novel approach to sample preparation for biomolecular SSNMR without crystallization or other sample manipulations. The biomolecules are confined by high gravity--obtained by centrifugal forces either directly in a SSNMR rotor or in a ultracentrifugal device--into a hydrated non-crystalline solid suitable for SSNMR investigations. When gravity is removed, the sample reverts to solution and can be treated as any solution NMR sample. We here describe a simple web tool to calculate the relevant parameters for the success of the experiment.

Great theoretical and methodological advances are pushing the limits of resolution and sensitivity in solid state NMR (SSNMR). However, sample preparation remains a critical issue for the success of an experiment. The factors affecting spectral quality in SSNMR samples are discussed, examining cases encountered in the literature and presenting new experimental data. A discussion on resolution and sensitivity in sedimented solutes is framed in this context.

Sedimented proteins have recently been shown to provide solid-state NMR spectra of high quality, suitable for structural investigation. This is ascribed to the strong self-crowding effect, which apparently increases the reorientation time up to the point that the protein can be considered immobile on the NMR timescale. Herein, a relaxometric investigation of sedimented bovine serum albumin is performed to obtain information on the dynamics of the system. The measurement of the proton longitudinal relaxation rates as a function of the applied magnetic field indicates that the sedimented protein has relaxation properties very different from those of the protein in concentrated water solutions, even in the presence of glycerol, and similar to those of slightly rehydrated lyophilized systems. This study confirms the hypothesis that the reorientation of the protein molecules is largely abolished in sediments., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have recently proposed sedimented solute NMR (SedNMR) as a solid-state method to access biomolecules without the need of crystallization or other sample manipulation. The drawback of SedNMR is that samples are intrinsically diluted and this is detrimental for the signal intensity. Ultracentrifugal devices can be used to increase the amount of sample inside the rotor, overcoming the intrinsic sensitivity limitation of the method. We designed two different devices and we here report the directions for using such devices and the relevant equations for determining the parameters for sedimentation.

The MaxOcc web portal is presented for the characterization of the conformational heterogeneity of two-domain proteins, through the calculation of the Maximum Occurrence that each protein conformation can have in agreement with experimental data. Whatever the real ensemble of conformations sampled by a protein, the weight of any conformation cannot exceed the calculated corresponding Maximum Occurrence value. The present portal allows users to compute these values using any combination of restraints like pseudocontact shifts, paramagnetism-based residual dipolar couplings, paramagnetic relaxation enhancements and small angle X-ray scattering profiles, given the 3D structure of the two domains as input. MaxOcc is embedded within the NMR grid services of the WeNMR project and is available via the WeNMR gateway at http://py-enmr.cerm.unifi.it/access/index/maxocc . It can be used freely upon registration to the grid with a digital certificate.

Multidomain proteins are composed of rigid domains connected by (flexible) linkers. Therefore, the domains may experience a large degree of reciprocal reorientation. Pseudocontact shifts and residual dipolar couplings arising from one or more paramagnetic metals successively placed in a single metal binding site in the protein can be used as restraints to assess the degree of mobility of the different domains. They can be used to determine the maximum occurrence (MO) of each possible protein conformation, i.e. the maximum weight that such conformations can have independently of the real structural ensemble, in agreement with the provided restraints. In the case of two-domain proteins, the metal ions can be placed all in the same domain, or distributed between the two domains. It has been demonstrated that the quantity of independent information for the characterization of the system is larger when all metals are bound in the same domain. At the same time, it has been shown that there are practical advantages in placing the metals in different domains. Here, it is shown that distributing the metals between the domains provides a tool for defining a coefficient of compatibility among the restraints obtained from different metals, without a significant decrease of the capability of the MO values to discriminate among conformations with different weights., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This perspective paper is intended to give some insights into the recently proposed technique of NMR of solutes sedimented by ultracentrifugation in a rotor used for solid state NMR experiments. Sedimented "states" correspond to molecules with very little reorientational capability in extremely concentrated solutions. They provide solid state NMR spectra comparable in quality with those of the best microcrystalline samples. Here we report some experiments to look for chemicals which affect the properties of the sediment, and we show that it is possible to fill the rotor in a true ultracentrifuge and then record the spectra. The latter possibility opens new horizons for NMR of sedimented systems.

Relatively large proteins in solution, spun in NMR rotors for solid samples at typical ultracentrifugation speeds, sediment at the rotor wall. The sedimented proteins provide high-quality solid-state-like NMR spectra suitable for structural investigation. The proteins fully revert to the native solution state when spinning is stopped, allowing one to study them in both conditions. Transiently sedimented proteins can be considered a novel phase as far as NMR is concerned. NMR of transiently sedimented molecules under fast magic angle spinning has the advantage of overcoming protein size limitations of solution NMR without the need of sample crystallization/precipitation required by solid-state NMR.

Differences between individual phenotypes are due both to differences in genotype and to exposure to different environmental factors. A fundamental contribution to the definition of the individual phenotype for clinical and therapeutic applications would come from a deeper understanding of the metabolic phenotype. The existence of unique individual metabolic phenotypes has been hypothesized, but the experimental evidence has been only recently collected. Analysis of individual phenotypes over the timescale of years shows that the metabolic phenotypes are largely invariant. The present work also supports the idea that the individual metabolic phenotype can also be considered a metagenomic entity that is strongly affected by both gut microbiome and host metabolic phenotype, the latter defined by both genetic and environmental contributions.

Since the recent availability of high sensitivity field-cycling relaxometers, it has become possible to measure the protein proton relaxation in millimolar protein solutions as a function of magnetic field. In principle, this provides direct access to the so-called spectral density function of protein protons and, hence, to a full set of dynamic parameters. Understanding the dynamic behavior of biological molecules is increasingly appreciated as crucial to understanding their function. However, theoretical tools to analyze the collective relaxation behavior of protons in solute macromolecules over a wide range of magnetic fields are lacking. A complete relaxation matrix analysis of such behavior is described here. This analysis provides excellent predictions of the experimental proton magnetization decays/recoveries-measured to an unprecedented level of accuracy by a last-generation fast field-cycling relaxometer-of two different globular proteins, hen egg white lysozyme and human serum albumin. The new experimentally validated theoretical model is then used to extract dynamic information on these systems. A "collective" order parameter SC2, different from, but complementary to, that commonly extracted from heteronuclear relaxation measurements at high field, is defined and measured. An accurate estimate of the rotational correlation time is obtained: in the case of lysozyme it agrees very well with theoretical predictions; in the case of serum albumin it provides evidence for aggregation at millimolar concentration.

Natively unfolded proteins are increasingly recognized to play important physiological roles. These proteins do not crystallize, so NMR is the only technique able to provide structural and dynamic information. However, in unfolded proteins, the proton chemical shift dispersion is poor, causing severe problems in resonance assignment. We designed a novel strategy based on two protonless experiments, a CBCACON-IPAP and a novel COCON-IPAP, that permits a straightforward and unequivocal backbone heteronuclear assignment of the natively unfolded protein alpha-synuclein.

We describe a new NMR experimental scheme that allows the direct determination of the dynamic frequency shift induced by chemical shift anisotropy/dipolar interaction (CSA/DD) cross-correlations in 15N-enriched proteins. Its principle consists of comparing two rates of polarisation transfer between the amide proton and nitrogen. The first rate, which is independent of the dynamic frequency shift, is based on a selective Hartmann-Hahn coherence transfer. The second rate, which depends on the dynamic frequency shift, is based on a free evolution of the transverse magnetisation. We report experimental validation of this approach by measuring the average dynamic frequency shift due to CSA/DD cross-correlations in the calcium-binding protein D9k. The method may also be applicable to the measurement of dynamic frequency shift induced by cross-correlations between the Curie spin and dipolar interactions.

Modules that use paramagnetism-based NMR restraints have been developed and integrated in the well known program for solution structure determination Xplor-NIH; the complete set of such modules is called PARArestraints for Xplor-NIH. Paramagnetism-based restraints are paramagnetic relaxation enhancements, pseudocontact shifts, residual dipolar couplings due to metal and overall magnetic anisotropy, and cross correlation between Curie relaxation and nuclear-nuclear dipolar relaxation. The complete program has been tested by back-calculating NOEs and paramagnetism-based restraints from the X-ray structure of cytochrome c (553) from B. pasteurii. Furthermore, the same experimental restraints previously used to determine the solution structure of cytochrome c (553) itself, of cytochrome b (5), and of calbindin D(9k) with the program PARAMAGNETIC DYANA, have been used for structure calculations by using PARArestraints for Xplor-NIH. The agreement between the two programs is quite satisfactory and validates both protocols.

This work presents the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002. Constraints are derived from homo- and heteronuclear one-, two- and three-dimensional (1)H and (15)N NMR data. Significant differences are outlined between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domain. In the NMR solution structure of PsaC the C-terminal region assumes a disordered helical conformation, and is clearly different from the extended coil conformation, which is one of the structural elements required to anchor PsaC to the PS I core heterodimer. In solution the N-terminus of PsaC is in contact with the pre-C-terminal region but slides in between the latter and the iron-sulfur core region of the protein. Together, these features result in a concerted movement of the N-terminus and pre-C-terminal region away from the F(A) binding site, accompanied by a bending of the N-terminus. In comparison, the same terminal regions are positioned much closer to F(A) and take up an anti-parallel beta-sheet arrangement in PsaC bound to PS I. The conformational changes between bound and unbound PsaC correlate with the differences reported earlier for the EPR spectra of reduced F(A) and F(B) in bound versus unbound PsaC. The observed different structural features in solution are highly relevant for unraveling the stepwise assembly process of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0321-3.

The full series of lanthanide ions (except the radioactive promethium and the S-state gadolinium) has been incorporated into the C-terminal calcium binding site of the dicalcium protein calbindin D(9k). A fairly constant coordination environment is maintained throughout the series. At variance with several lanthanide complexes with small chelating ligands investigated in the past, the large protein moiety provides a large number of NMR signals whose hyperfine shifts can be exclusively ascribed to pseudocontact shifts (PCS). The chemical shifts of 1H and 15N backbone and side chain amide NH groups were accurately measured through HSQC experiments. 1097 PCS were estimated from these by subtracting the diamagnetic contributions measured on HSQC spectra of either the 4f(0) lanthanum(III) or the 4f(14) lutetium(III) derivatives and used to define a quality factor for the structure. The differences in diamagnetic chemical shifts between the two diamagnetic blanks were relatively small, although some were not negligible especially for the nuclei closest to the metal center. These differences were used as a tolerance for the PCS. The magnetic susceptibility tensor anisotropies for each paramagnetic lanthanide ion were obtained as the result of the solution structure determination performed by using the NOEs of the cerium(III) derivative and the PCS of all lanthanides simultaneously. This set of reliable magnetic data permits an experimental assessment of Bleaney's theory relative to the magnetic properties for an extended series of lanthanide complexes in solution. All of the obtained tensors show some rhombicity, as could be expected from the lack of symmetry of the protein environment. The directions of the largest magnetic susceptibility component for Ce, Pr, Nd, Sm, Tb, Dy, and Ho and of the smallest magnetic susceptibility component for Eu, Er, Tm, and Yb were found to be all within 15 degrees from their average (within 20 degrees for Sm), confirming the essential similarity of the coordination environment for all lanthanides. Bleaney's theory is in excellent qualitative agreement with the observed pattern of axial anisotropies. Its quantitative agreement is substantially better than that suggested by previous analyses performed on more limited sets of PCS data for small lanthanide complexes, the so-called crystal field parameter varying only within +/-30% from one lanthanide to another. These variations are even smaller (+/-15%) if a reasonable T(-3) correction is taken into consideration. A knowledge of magnetic susceptibility anisotropy properties of lanthanides is essential in determining the self-orienting properties of lanthanide complexes in solution when immersed in magnetic fields.

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Recombinant PsaC was reconstituted in vitro and investigated by UV/vis, EPR, and 1H NMR spectroscopy. Its UV/vis and EPR spectroscopic properties correspond to those of the wild-type protein. Fast repetition 1D and 2D 1H NMR spectra allowed the sequence-specific assignment of the hyperfine-shifted proton resonances of the cluster-ligating resonances, taking advantage also of chemical shift analogies with other 4 and 8 Fe ferredoxins and a structural model for PsaC. The Calpha-Cbeta-S-Fe dihedral angles of the cluster ligands could be estimated from the chemical shifts and relaxation properties of their betaCH2 protons. All NMR-derived structural information on PsaC confirms its similarity to smaller 8Fe ferredoxins serving as electron transfer proteins in solution. Partial reduction of PsaC leads to an intermediate species with strongly exchange broadened 1H NMR resonances. The intermolecular electron exchange rate is estimated to be in the 10(2)-10(4) s-1 range, the intramolecular electron exchange rate between the two [Fe4S4] clusters to be higher than 10(4) s-1. The consequences of these findings for the electron transfer in photosystem I are discussed.