Publishing 13C metabolic flux analysis studies: A review and future perspectives

¹³C-Metabolic flux analysis (¹³C-MFA) is a powerful model-based analysis technique for determining intracellular metabolic fluxes in living cells. It has become a standard tool in many labs for quantifying cell physiology, e.g., in metabolic engineering, systems biology, biotechnology, and biomedical research. With the increasing number of ¹³C-MFA studies published each year, it is now ever more important to provide practical guidelines for performing and publishing ¹³C-MFA studies so that quality is not sacrificed as the number of publications increases. The main purpose of this paper is to provide an overview of good practices in ¹³C-MFA, which can eventually be used as minimum data standards for publishing ¹³C-MFA studies. The motivation for this work is two-fold: (1) currently, there is no general consensus among researchers and journal editors as to what minimum data standards should be required for publishing ¹³C-MFA studies; as a result, there are great discrepancies in terms of quality and consistency; and (2) there is a growing number of studies that cannot be reproduced or verified independently due to incomplete information provided in these publications. This creates confusion, e.g. when trying to reconcile conflicting results, and hinders progress in the field. Here, we review current status in the ¹³C-MFA field and highlight some of the shortcomings with regards to ¹³C-MFA publications. We then propose a checklist that encompasses good practices in ¹³C-MFA. We hope that these guidelines will be a valuable resource for the community and allow ¹³C-flux studies to be more easily reproduced and accessed by others in the future.     

Stereochemistry of peptides containing 1-aminocyclopentanecarboxylic acid (Acc5): Solution and solid-state conformations of Boc-Acc5-Acc5-NHMe

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid (Acc⁵) has been carried out. ¹H-nmr studies establish a β-turn conformation for Boc-Acc⁵-Acc⁵-NHMe in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III β-turn conformation in the solid state stabilized by a 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The ?,ψ values for both Acc⁵ residues are close to those expected for an ideal 3₁₀-helical conformation (?? ± 60°, ψ～ ±30°).     

Site-selective <Superscript>13</Superscript>C labeling of proteins using erythrose

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with ¹³C and/or ¹H, which is achieved in the most general way by using site-selectively ¹³C-enriched glucose (1- and 2-¹³C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively ¹³C-enriched erythrose (1-, 2-, 3- and 4-¹³C) as a suitable precursor for ¹³C labeled aromatic side chains. We quantify ¹³C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the ¹³C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated ¹³C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective ¹³C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.     

Structural Studies of Bcl-xL/ligand Complexes using 19F NMR

Fluorine atoms are often incorporated into drug molecules as part of the lead optimization process in order to improve affinity or modify undesirable metabolic and pharmacokinetic profiles. From an NMR perspective, the abundance of fluorinated drug leads provides an exploitable niche for structural studies using ¹⁹F NMR in the drug discovery process. As ¹⁹F has no interfering background signal from biological sources, ¹⁹F NMR studies of fluorinated drugs bound to their protein receptors can yield easily interpretable and unambiguous structural constraints. ¹⁹F can also be selectively incorporated into proteins to obtain additional constraints for structural studies. Despite these advantages, ¹⁹F NMR has rarely been exploited for structural studies due to its broad lines in macromolecules and their ligand complexes, leading to weak signals in ¹H/¹⁹F heteronuclear NOE experiments. Here we demonstrate several different experimental strategies that use ¹⁹F NMR to obtain ligand–protein structural constraints for ligands bound to the anti-apoptotic protein Bcl-xL, a drug target for anti-cancer therapy. These examples indicate the applicability of these methods to typical structural problems encountered in the drug development process.     

68Ga‐labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators

The dual interaction with integrins and neuropilin‐1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with ⁶⁸GaCl₃. Negatively charged ⁶⁸Ga‐DOTAGA‐iRGD and neutral ⁶⁸Ga‐NODAGA‐iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral ⁶⁸Ga‐NODAGA‐iRGD, but ⁶⁸Ga‐DOTAGA‐iRGD exhibited better tumor‐to‐blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.     

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5′-AGGG(TTAGGG)₃] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na⁺ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K⁺ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K⁺ solutions. Sedimentation coefficients were determined experimentally in both Na⁺ and K⁺ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na⁺ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K⁺ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K⁺ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K⁺ solutions, but not in Na⁺ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K⁺ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K⁺ solution is not the one determined in the published crystalline state.     

Protein <Superscript>19</Superscript>F-labeling using transglutaminase for the NMR study of intermolecular interactions

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic ¹⁵N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. ¹⁹F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, ¹⁹F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic ¹⁹F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The ¹⁹F-labeling method was 3.5-fold more sensitive than ¹⁵N-labeling, and could be combined with other chemical modification techniques such as lysine ¹³C-methylation. ¹³C-dimethylated-¹⁹F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.     

Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Cholesterol as a Risk Factor for Subarachnoid Hemorrhage: A Systematic Review

BackgroundThe role played by total cholesterol (TC) in risk for subarachnoid hemorrhage (SAH) is unclear because studies report both high and low TC each as a risk factor. We performed a systematic review to clarify associations between lipid profile and SAH.MethodsOur literature search comprised Pubmed, Scopus, and Cochrane Library databases with no language, publication year, or study type limitations. The Preferred Reporting Items for Systematic reviews and Meta-analyses (PRISMA) checklist guided our reporting. Data forms adapted from the Critical Appraisal Skills Program (CASP), and Cochrane Collaboration guidelines provided a platform for risk-of-bias evaluation. We used a random effects model to calculate pooled estimates and assessed heterogeneity with I²-statistics.ResultsOf the final 21 studies reviewed, 12 were prospective and 9 retrospective. All studies assessed TC, four assessed HDL, and none LDL in risk for SAH. Heterogeneity among all, retrospective, and Asian studies was high (I² = 79.5%, I² = 89.0%, and I² = 84.3%) and considerable in prospective (I² = 46.0%). We therefore focused on qualitative analysis and found that only two studies had a low risk of bias. According to these studies high TC increases risk for SAH in men, whereas the role of HDL remained unclear.ConclusionThe low-risk-of-bias studies suggest that elevated TC levels elevate risk for SAH in men. Due to the high prevalence of hypercholesterolemia, population attributable risk (PAR) of hypercholesterolemia may exceed the PARs of smoking and hypertension in men. Apart from diabetes and obesity, the risk-factor profile of SAH seems to resemble that of other cerebrovascular diseases, at least in men.     

Characterization of Mg2+-ATPase from sheep kidney medulla: magnetic resonance and kinetic studies.

Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg²⁺ or Mn²⁺ indicate a biphasic response to divalent cations. Apparent K_m values of 23 μm for free Mg²⁺ and 3.3 μm for free Mn²⁺ are obtained at low levels of added metal, while K_m values of 0.50 mm for free Mg²⁺ and 0.43 mm for free Mn²⁺ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg²⁺-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent K_m values for free ATP of 6.0 and 1.4 μm with Mg²⁺ and Mn²⁺, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that K_m values for free ATP of 8.0 and 2.0 mm are obtained for Mg²⁺ and Mn²⁺, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg²⁺-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with K_i values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn²⁺ per 469,000 g of protein. The Mn²⁺ binding studies yield a K_D for Mn²⁺ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn²⁺ at low Mn²⁺ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn²⁺ per single high affinity Mn²⁺ site. The K_D for Mn²⁺ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn²⁺ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn²⁺ in the presence of the enzyme is also consistent with the tight binding of a single Mn²⁺ ion per 469,000 M_r protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn²⁺ at the single tight site, ?_b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn²⁺ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10^?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg²⁺-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg²⁺-ATPase described here are discussed.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

High-affinity binding of 3H-imipramine in brain and platelets and its relevance to the biochemistry of affective disorders

Specific high-affinity binding of ³H-imipramine has been demonstrated in the brain of various species including man. These binding sites have many of the characteristics to be expected for a pharmacological receptor and appear to be associated with the neuronal uptake mechanism for serotonin. Different antidepressant treatments like chronic administration of tricyclic antidepressants, chronic electroshock or sleep-deprivation result in decreases in the density of ³H-imipramine binding sites in normal animals. ³H-imipramine binding sites have also been found in blood platelets from different species including man. These sites are identical to those described in the brain. Clinical studies have shown that untreated severely depressed patients have a lower density of ³H-imipramine binding sites in their platelets when compared with control volunteers of the same age and sex. Longitudinal studies indicate that the low density of ³H-imipramine binding sites does not change during treatment with tricyclic antidepressant drugs and the subsequent clinical recovery from depression. ³H-imipramine binding in brain and platelets is proposed as a useful research tool in biochemical and clinical studies in affective disorders.     

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34^? is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34^? HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34⁺ bone marrow cells as immunogen. Thus, neither MSC being CD34^? nor HSC being CD34⁺ is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34⁺ fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.     

A highly sensitive method for the estimation of pyrimidine dimers in DNA by high-pressure liquid cation-exchange chromatography

We have developed a rapid, highly sensitive method for the separation of thymine-dimers from thymine on a cation-exchange resin, which is capable of detecting one dimer out of 5 × 10³ to 10⁴ thymine molecules (depending on the specific activity of the used material) by the means of high-pressure liquid chromatography. The assay has been very useful in DNA-hybridization studies using $\text{(}\text{TT}\text{)}$-containing DNA-strands and there is evidence that the method will be valuable for photoreactivation studies of uv-damaged DNA and for the enzymes involved in dimer-excision studies.     

In silico, NMR and pharmacological evaluation of an hydroxyoxindole cholinesterase inhibitor

From a screening study of various potential inhibitors for cholinesterases (ChEs), compound (rac)-1 (4-((3-hydroxy-2-oxo-3-phenylindolin-1-yl) methyl) piperidin-1-ium chloride) showed an IC₅₀ of 18?μM for butyrylcholinesterase (BuChE). Herein we present a toxicological and pharmacological evaluation of (rac)-1 to determine its potential for use as an alternative ChE inhibitor for the treatment of Alzheimer’s disease. The strategy adopted included in vivo and ex vivo studies with mouse models, Molecular Modelling and Saturation Transfer Difference (STD) NMR studies.Preliminary molecular docking studies were conducted with both (R) and (S)-1 with acetylcholinesterase (AChE) and BuChE, prior to advancing to the mouse model, and indeed favorable interactions were observed, with (R)-1 showing the best binding with AChE and (S)-1 with BuChE. STD-NMR studies were used to successfully validate these results. Toxicological studies were also conducted using the Artemia salina model, with donepezil as reference. It was found that in the in vivo mouse studies that (rac)-1 presented a slightly better inhibition of AChE (0.096?µmol.min^?1.mg^?1) than donepezil (0.112?µmol.min^?1.mg^?1) and the same level of inhibition for BuChE as donepezil (0.014?µmol.min^?1.mg^?1).     

Interactions of DNA with divalent metal ions. IV. Competitive studies of Mn2+ binding to AT- and GC-rich DNAs

It has been inferred from previous studies that Mn²⁺ ions bind preferentially to G·C base pairs in DNA, and it has even been suggested that this preference for G·C pairs might be responsible for some of the Mn²⁺ specific effects observed in various biochemical reactions. In this paper we investigate the AT/GC preference of Mn²⁺ by direct competition studies in which AT-rich DNA was dialyzed against GC-rich DNA in the presence of varying amounts of Mn²⁺. Analysis of these results demonstrates that over a wide range of Mn²⁺/DNA(P) molar ratios, Mn²⁺ binds to A·T and to G·C base pairs with virtually identical affinity, although in a somewhat different mode. Both the present and previous nmr, uv, CD, and melting studies are discussed in terms of the different modes of binding of Mn²⁺ to single- and double-stranded DNA.     

Interaction of MgATP2− with DNA: Assessment of metal binding sites and DNA conformations by spectroscopic and thermal denaturation studies

Spectroscopic (IR, UV, CD and fluorescence) and thermal denaturation studies of native calf thymus DNA, DNAMgATP²⁻ and DNAMg²⁺ have been carried out in aqueous KBr medium (introduced by the present authors as a very effective solvent for DNA). The IR data recorded for the systems indicate that MgATP²⁻ binds to the N₇ and C₆O of the guanine residue of DNA forming a five-membered chelate ring. The data also suggest that despite binding to the guanine bases, Mg²⁺ binds more strongly to the phosphate moiety of DNA. Solution CD spectra of DNA, DNAMgATP²⁻ and DNAMg²⁺ indicate that in each case DNA exists in the B conformation. Thin-film CD studies reveal that irrespective of the relative humidity conditions, pure DNA as well as that after interaction with Mg²⁺ show a structural transition B → C, conformationally, although belonging to the B family. A similar study shows that DNA on interaction with MgATP²⁻ assumes a more packed conformation (B)_n giving rise to a ψ⁻ spectrum. Steady-state as well as dynamic fluorimetric studies clearly indicate that MgATP²⁻ does not intercalate between CGGC base pairs. The thermal denaturation studies support the IR data with respect to the metal binding sites and the mode of binding in both cases.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Pituitary localization of 3H-spiroperidol by an Uptake/Storage Mechanism?

The lack of a pituitary imaging agent combined with the considerable clinical value for such an agent prompted an examination of ³H-spiroperidol (³HSp). Spiroperidol was selected for initial evaluation based on its high affinity for D₂ receptors which are known to be present in the pituitary. A time course study of ³HSp concentration in rat pituitary and other tissues was conducted. Pituitary activity levels were found to be constant from 5 min to 4 h and were about 8 times levels in corpus striatum at 1 h. Blocking studies with (+)-butaclamol and with unlabelled spiroperidol suggested the existence of both a D₂ receptor mediated binding localization and a second uptake which is postulated to be an internalization process. Further studies involving ultracentrifugation of pituitary homogenates resulted in evidence for association of ³HSp with dense subcellular particles. ³HSp thus appears to be internalized by pituitary cells.     

Structural Analysis of Divalent Metals Binding to the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Response Regulator Spo0F: The Possibility for <Emphasis Type="Italic">In Vitro</Emphasis> Metalloregulation in the Initiation of Sporulation

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and μESI-MS. NMR studies show that the divalent metals Ca²⁺, Mg²⁺ and Mn²⁺ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu²⁺ show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the β4-α4 loop and the β5/α5 interface, particularly around and including H101. μESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu²⁺, multiple metal-bound species are observed. Subsequent experiments reveal that Mg²⁺ supports phosphotransfer between KinA and Spo0F, while Cu²⁺ fails to support KinA phosphotransfer. Additionally, the presence of Cu²⁺ at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.