Position-specific carbon stable isotope analysis of glyphosate: isotope fingerprinting of molecules within a mixture.

Glyphosate [N-(phosphonomethyl) glycine] is a widely used herbicide and a molecule of interest in the environmental sciences, due to its global use in agriculture and its potential impact on ecosystems. This study presents the first position-specific carbon isotope (13C/12C) analyses of glyphosates from multiple sources. In contrast to traditional isotope ratio mass spectrometry (IRMS), position-specific analysis provides 13C/12C ratios at individual carbon atom positions within a molecule, rather than an average carbon isotope ratio across a mixture or a specific compound. In this work, glyphosate in commercial herbicides was analyzed with only minimal purification, using a nuclear magnetic resonance (NMR) spectroscopy method that detects 1H nuclei with bonds to either 13C or 12C, and isolates the signals of interest from other signals in the mixture. Results demonstrate that glyphosate from different sources can have significantly different intramolecular 13C/12C distributions, which were found to be spread over a wide range, with δ13C Vienna Peedee Belemnite (VPDB) values of -28.7 to -57.9‰. In each glyphosate, the carbon with a bond to the phosphorus atom was found to be depleted in 13C compared to the carbon at the C2 position, by 4 to 10‰. Aminomethylphosphonic acid (AMPA) was analyzed for method validation; AMPA contains only a single carbon position, so the 13C/12C results provided by the NMR method could be directly compared with traditional isotope ratio mass spectrometry. The glyphosate mixtures were also analyzed by IRMS to obtain their average 13C/12C ratios, for comparison with our position-specific results. This comparison revealed that the IRMS results significantly disguise the intramolecular isotope distribution. Finally, we introduce a 31P NMR method that can provide a position-specific 13C/12C ratio for carbon positions with a C-P chemical bond, and the results obtained by 1H and 31P for C3 carbon agree with one another within their analytical uncertainty. These analytical tools for position-specific carbon isotope analysis permit the isotopic fingerprinting of target molecules within a mixture, with potential applications in a range of fields, including the environmental sciences and chemical forensics.

Novel Nuclear Magnetic Resonance Method for Position-Specific Carbon Isotope Analysis of Organic Molecules with Significant Impurities.

We introduce a novel nuclear magnetic resonance (NMR) tool for determining position-specific carbon (13C/12C) isotope ratios within complex organic molecules. This analytical advancement allows us to measure position-specific isotope ratios of samples that contain impurities with NMR peaks that overlap with the signals of interest. The method involves collecting a series of alternating 13C-coupled and 13C-decoupled 1H NMR spectra using an NMR pulse sequence designed to optimize temperature stability, followed by a data reduction scheme that allows the signals of interest to be isolated from signals of impurities. The method was validated using glycine reference materials with known 13C/12C ratios from the US Geological Survey (USGS) into which impurities typically found in amino acid samples were intentionally introduced. Following validation, the method was used to determine position-specific 13C/12C ratios in a set of USGS l-valine materials (USGS73, -74, -75) that contain significant impurities associated with their biological origin. The l-valines were found to contain distinct intramolecular isotope variability, and the 13Cα isotope spikes in USGS74 and USGS75 were clearly detected, where they preserve carbon isotope ratios of -4.8 ± 0.9‰ and +11.5 ± 0.8‰, respectively. Carbon isotope abundance at the beta and gamma positions indicates that the USGS73 l-valine was obtained from a different source than USGS74 and -75. This analytical approach is a significant step forward in the field of position-specific isotope analysis at natural abundance via NMR because it enables the investigation of samples that contain impurities which are typically present in samples derived from natural sources.

Absolute Carbon Stable Isotope Ratio in the Vienna Peedee Belemnite Isotope Reference Determined by 1H NMR Spectroscopy.

The Vienna Peedee Belemnite (VPDB) isotope reference defines the zero point of the carbon stable isotope scale that is used to describe the relative abundance of 13C and 12C. An accurate and precise characterization of this isotope reference is valuable for interlaboratory comparisons and conducting robust carbon stable isotope analyses in a vast array of fields, such as chemical forensics, (bio)geochemistry, ecology, or (astro)biology. Here, we report an absolute 13C/12C ratio for VPDB that has been obtained, for the first time, using proton nuclear magnetic resonance spectroscopy (1H NMR). Four different NMR instruments were used to determine 13C/12C ratios in a set of glycine reference materials from the US Geological Survey (USGS64, USGS65, and USGS66) and a set of formate samples that were characterized by isotope ratios mass spectrometry (IRMS). Intercalibration of the NMR-derived 13C/12C ratios with relative abundance (δ13CVPDB) measurements from IRMS yields a value of 0.011100 for the absolute 13C/12C ratio in VPDB, with an expanded uncertainty of ±0.000026 (2σ, n = 114). This is significantly different from the value of 0.011180 that is commonly used but falls within the range of values recently revised using IRMS and infrared absorption measurements. 1H NMR was found to be an effective method for measuring absolute 13C/12C ratios due to its ability to simultaneously detect signals associated with 12C and 13C. Results provide a new and independent measure of the carbon isotope composition of VPDB, improving our understanding of this important isotope reference.

Intramolecular distribution of 13C/12C isotopes in amino acids of diverse origins.

Carbon stable isotope analysis can provide information about the origin and synthetic pathways that produce organic molecules, with applications in chemical, medical and (bio)geochemical sciences. The 13C/12C isotope ratios of organics such as amino acids are most commonly obtained as whole molecule averages. In this study, we apply proton nuclear magnetic resonance spectroscopy to conduct position-specific carbon isotope analyses of L-/D-alanine, L-threonine and L-histidine from different sources, in addition to molecule average stable isotope analyses obtained via mass spectrometry. Our results demonstrate that carbon isotope ratios can vary significantly between the individual carbon positions within an amino acid. For example, the ß- and γ- carbons of L-threonine can differ in 13C/12C ratio by > 20 ‰. Comparisons of the position-specific and whole molecule average stable isotope abundances show that whole molecule analyses can mask the intramolecular isotope variation. These results provide the first experimentally measured position-specific isotope ratios for alpha and side chain carbons of alanine, threonine and histidine. Comparison with previous ab initio calculations of intramolecular equilibrium fractionation shows that the carbon isotope distributions are not at equilibrium, thus kinetic isotope effects play a significant role in amino acid synthesis. We hypothesize that position-specific 13C/12C isotope ratios provide an "isotopic fingerprint" that can give insight into the origin or synthesis pathway that formed an amino acid, and that this emerging analytical field will be a valuable addition to traditional stable isotope analysis.

Position-Specific Carbon Stable Isotope Ratios by Proton NMR Spectroscopy.

Carbon stable isotopes provide insights into the origin and synthesis pathway of an organic molecule, and hence, contribute information that is fundamental to understanding chemical, physiological, and ecological processes. Organic carbon 13C/12C isotope ratios are commonly obtained as whole-molecule averages or as measurements of bulk samples. In contrast, position-specific isotope analysis (PSIA) provides isotope ratios for the individual carbons within a molecule, providing additional information that is masked by traditional analytical techniques. Here we introduce a 1H NMR method for determining position-specific 13C/12C ratios within organic molecules. A peak shape superposition procedure is used to bypass the need for traditional peak integration, by exploiting relationships among the shapes of 1H and 13C satellite peaks in 1H NMR spectra. The method also has a significant sensitivity advantage over NMR methods that utilize direct detection of 13C. Furthermore, we demonstrate that isotope standard materials (such as those obtainable from U.S. Geological Survey) are indispensable in calibrating an NMR instrument, in order to obtain accurate isotope ratio results. Our analytical approach was applied to organic molecules of different complexity and origin, including ethanols, propionic acids, and thymidine. Results verify that chemically identical molecules from different sources can have different intramolecular isotope distributions; hence position-specific 13C/12C ratios provide an isotopic fingerprint of an organic molecule. Position-specific information for the nucleoside thymidine, where five of eight carbon positions were measured, is significant because its complexity would make it a difficult target for PSIA by mass spectrometry. The 1H NMR method is complementary to other methods of PSIA, and will make 13C/12C PSIA employable to a wider range of organic molecules.

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

Methylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwise sequence identity (35%) among the isozyme pairs in the parallel pathways, and could offer insight into substrate preferences and pathway functions. The kinetic parameters of tautomerase I and tautomerase II were determined using 2-hydroxymuconate and 5-(methyl)-2-hydroxymuconate. Both tautomerase I and tautomerase II process the substrates, but with different efficiencies. Crystal structures were determined for both tautomerase I and tautomerase II, at 1.57 and 1.64Å resolution, respectively. The backbones of tautomerase I and tautomerase II are highly similar, but have distinct active site environments. The results, in combination with those for other structurally and kinetically characterized 4-OT isozymes, suggest that tautomerase I catalyzes the tautomerization of both 2-hydroxymuconate and alkyl derivatives, whereas tautomerase II might specialize in other aromatic hydrocarbon metabolites.

The missing linker: a dimerization motif located within polyketide synthase modules.

The dimerization of multimodular polyketide synthases is essential for their function. Motifs that supplement the contacts made by dimeric polyketide synthase enzymes have previously been characterized outside the boundaries of modules, at the N- and C-terminal ends of polyketide synthase subunits. Here we describe a heretofore uncharacterized dimerization motif located within modules. The dimeric state of this dimerization element was elucidated through the 2.6 Å resolution crystal structure of a fragment containing a dimerization element and a ketoreductase. The solution structure of a standalone dimerization element was revealed by nuclear magnetic resonance spectroscopy to be consistent with that of the crystal structure, and its dimerization constant was measured through analytical ultracentrifugation to be ∼20 µM. The dimer buries ∼990 Å(2) at its interface, and its C-terminal helices rigidly connect to ketoreductase domains to constrain their locations within a module. These structural restraints permitted the construction of a common type of polyketide synthase module.

Bruno protein contains an expanded RNA recognition motif.

The RNA recognition motif (or RRM) is a ubiquitous RNA-binding module present in approximately 2% of the proteins encoded in the human genome. This work characterizes an expanded RRM, which is present in the Drosophila Bruno protein, and targets regulatory elements in the oskar mRNA through which Bruno controls translation. In this Bruno RRM, the deletion of 40 amino acids prior to the N-terminus of the canonical RRM resulted in a significantly decreased affinity of the protein for its RNA target. NMR spectroscopy showed that the expanded Bruno RRM contains the familiar RRM fold of four antiparallel beta-strands and two alpha-helices, preceded by a 10-residue loop that contacts helix alpha(1) and strand beta(2); additional amino acids at the N-terminus of the domain are relatively flexible in solution. NMR results also showed that a truncated form of the Bruno RRM, lacking the flexible N-terminal amino acids, forms a stable and complete canonical RRM, so that the loss of RNA binding activity cannot be attributed to disruption of the RRM fold. This expanded Bruno RRM provides a new example of the features that are important for RNA recognition by an RRM-containing protein.

A pseudocatenane structure formed between DNA and A cyclic bisintercalator.

Targeting double-stranded DNA with small molecules remains an active area of basic research. Herein is described a cyclic DNA bisintercalator that is based on two naphthalene diimide (NDI) intercalating units tethered by one linking element specific for binding in the minor groove and the other linking element specific for binding in the major groove. DNase I footprinting revealed a strong preference for binding the sequence 5'-GGTACC-3'. NMR structural studies of the complex with d(CGGTACCG)(2) verified a pseudocatenane structure in which the NDI units reside four base pairs apart, with one linker segment located in the minor groove and the other in the major groove consistent with the linker designs. To the best of our knowledge, this is the first structurally well-characterized pseudocatenane complex between a sequence specific cyclic bisintercalator and intact DNA.

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis.

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.

Domain C of human poly(ADP-ribose) polymerase-1 is important for enzyme activity and contains a novel zinc-ribbon motif.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins using NAD (+) as a substrate. In this work, NMR methods were used to determine the solution structure of human PARP-1 protein. Domain C was found to contain a zinc-binding motif of three antiparallel beta-strands with four conserved cysteines positioned to coordinate the metal ligand, in addition to a helical region. The zinc-binding motif is structurally reminiscent of the "zinc-ribbon" fold, but with a novel spacing between the conserved cysteines (CX2CX12CX 9C). Domain C alone does not appear to bind to DNA. Interestingly, domain C is essential for PARP-1 activity, since a mixture containing nicked DNA and the PARP-1 ABDEF domains has only basal enzymatic activity, while the addition of domain C to the mixture initiated NAD (+) hydrolysis and the formation of poly(ADP-ribose), as detected by an NMR-based assay and autoradiography. The structural model for domain C in solution provides an important framework for further studies aimed at improving our understanding of how the various domains within the complex PARP-1 enzyme play their respective roles in regulating the enzyme activity when cells are under conditions of genotoxic stress.

The structure of eukaryotic translation initiation factor-4E from wheat reveals a novel disulfide bond.

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m(7) guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight beta-strands, three alpha-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m(7)GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m(7)GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m(7)GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s(-1).

Structural characterization of a rigidified threading bisintercalator.

NMR spectroscopy was used to explore the sequence-specific interaction of DNA with a new threading bisintercalator (C1) consisting of two intercalating 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) units connected by a rigid, tricyclic spiro linker. A structural model of C1 complexed to d(CGGTACCG)(2) was calculated using distance constraints obtained from solution NMR data. The model was also supported by the results from residual dipolar coupling (RDC) measurements obtained using Pf1-phage as a cosolvent. According to the model, the central cyclohexane ring of the linker connecting the two NDI units lies flat in the minor groove of DNA. Linker length, hydrogen bonding, steric, and hydrophobic factors all appear to contribute to the observed sequence specificity of binding. These results serve to illustrate the versatility of threading polyintercalation given that, in a previous study, a ligand consisiting of two NDI units joined by a flexible peptide linker was shown to bind sequence specifically within the major groove of this same sequence of DNA.

Global metabolic changes following loss of a feedback loop reveal dynamic steady states of the yeast metabolome.

Metabolic enzymes control cellular metabolite concentrations dynamically in response to changing environmental and intracellular conditions. Such real-time feedback regulation suggests the global metabolome may sample distinct dynamic steady states, forming "basins of stability" in the energy landscape of possible metabolite concentrations and enzymatic activities. Using metabolite, protein and transcriptional profiling, we characterize three dynamic steady states of the yeast metabolome that form by perturbing synthesis of the universal methyl donor S-adenosylmethionine (AdoMet). Conversion between these states is driven by replacement of serine with glycine+formate in the media, loss of feedback inhibition control by the metabolic enzyme Met13, or both. The latter causes hyperaccumulation of methionine and AdoMet, and dramatic global compensatory changes in the metabolome, including differences in amino acid and sugar metabolism, and possibly in the global nitrogen balance, ultimately leading to a G1/S phase cell cycle delay. Global metabolic changes are not necessarily accompanied by global transcriptional changes, and metabolite-controlled post-transcriptional regulation of metabolic enzymes is clearly evident.

Solution structure of a conserved domain of antizyme: a protein regulator of polyamines.

Antizyme and its isoforms are members of an unusual yet broadly conserved family of proteins, with roles in regulating polyamine levels within cells. Antizyme has the ability to bind and inhibit the enzyme ornithine decarboxylase (ODC), targeting it for degradation at the proteasome; antizyme is also known to affect the transport of polyamines and interact with the antizyme inhibitor protein (AZI), as well as the cell-cycle protein cyclin D1. In the present work, NMR methods were used to determine the solution structure of a stable, folded domain of mammalian antizyme isoform-1 (AZ-1), consisting of amino acid residues 87-227. The protein was found to contain eight beta strands and two alpha helices, with the strands forming a mixed parallel and antiparallel beta sheet. At the level of primary sequence, antizyme is not similar to any protein of known structure, and results show that antizyme exhibits a novel arrangement of its strands and helices. Interestingly, however, the fold of antizyme is similar to that found in a family of acetyl transferases, as well as translation initiation factor IF3, despite a lack of functional relatedness between these proteins. Structural results, combined with amino acid sequence comparisons, were used to identify conserved features among the various homologues of antizyme and their isoforms. Conserved surface residues, including a cluster of acidic amino acids, were found to be located on a single face of antizyme, suggesting this surface is a possible site of interaction with target proteins such as ODC. This structural model provides an essential framework for an improved future understanding of how the different parts of antizyme play their roles in polyamine regulation.

The hydratase activity of malonate semialdehyde decarboxylase: mechanistic and evolutionary implications.

Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic beta-alpha-beta-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a kcat/Km value of 6.0 x 102 M-1 s-1. Pro-1 and Arg-75 are critical for both activities, and the pKa of Pro-1 was determined to be approximately 9.2 by a direct 15N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pKa value of Pro-1 are shared with trans-3-chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water.

Crystal structure of archaeal ribonuclease P protein aRpp29 from Archaeoglobus fulgidus.

The crystal structure of ribonuclease P protein aRpp29 from the sulfate-reducing hyperthermophile Archaeoglobus fulgidus was determined at 1.7 A resolution using X-ray diffraction methods. The central feature of this archaeal protein is a sheet of six antiparallel beta-strands twisted around a conserved hydrophobic core. Residues near the N- and C-termini form helical structures that are oriented in an antiparallel manner. A comparison of conserved amino acids indicates that archaeal aRpp29 is homologous to human ribonuclease P protein Rpp29. The aRpp29 protein is structurally similar to bacterial transcription factors Hfq and NusG, as well as the Sm and Sm-like RNA-associated proteins from eukarya. The crystal structure of A. fulgidus aRpp29 differs from the previously reported solution structure, where NMR data did not detect the helices and indicated that approximately 40% of the residues are relatively flexible or disordered. Circular dichroism data indicate that the protein has less helical content than the amount observed in the crystal, suggesting that in solution the helical regions are unfolded or in equilibrium between folded and unfolded forms; this hypothesis is consistent with amide proton exchange rate data. Surface residues that are conserved from archaea to humans and are likely to interact with the ribonuclease P RNA or other protein subunits are identified in the structure. The model of the aRpp29 protein defined by this work provides an essential step toward eventually understanding the overall architecture of ribonuclease P.

NMR structural analysis of a modular threading tetraintercalator bound to DNA.

The synthesis and NMR structural studies are reported for a modular threading tetraintercalator bound to DNA. The tetraintercalator design is based on 1,4,5,8-tetracarboxylic naphthalene diimide units connected through flexible peptide linkers. Aided by an overall C(2) symmetry, NMR analysis verified a threading polyintercalation mode of binding, with linkers alternating in the order minor groove, major groove, minor groove, analogous to how a snake might climb a ladder. This study represents the first NMR analysis of a threading tetraintercalator and, as such, structurally characterizes a new topology for molecules that bind to relatively long DNA sequences with extensive access to both DNA grooves.

The N-terminal domain (IF2N) of bacterial translation initiation factor IF2 is connected to the conserved C-terminal domains by a flexible linker.

Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA. Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods. Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein. (15)N NMR relaxation rate data indicate that (1) the IF2N domain is internally well ordered and tumbles in solution in a manner that is independent of the other domains of the IF2 protein, and (2) the IF2N domain is connected to the C-terminal regions of IF2 by a flexible linker. Chemical shifts of resonances within the isolated IF2N domain do not significantly differ from those of the corresponding residues within the context of the full-length 97-kD protein, indicating that IF2N is a structurally independent unit that does not strongly interact with other regions of IF2. CD and NMR data together provide evidence that Domains I-III of IF2 have unstructured and flexible regions as well as substantial helical content; CD data indicate that the helical content of these regions decreases significantly at temperatures above 35 degrees C. The features of structurally well-ordered N- and C-terminal domains connected by a flexible linker with significant helical content are reminiscent of another translation initiation factor, IF3.

NMR structure of an archaeal homologue of ribonuclease P protein Rpp29.

A protein component of the Archaeoglobus fulgidus RNase P was expressed in Escherichia coli, purified, and structurally characterized using multidimensional NMR methods. The dominant structural feature of this 11 kDa protein is a sheet of six antiparallel beta-strands, wrapped around a core of conserved hydrophobic amino acids. Amide proton exchange and (15)N relaxation rate data provide evidence that the first 16 residues of the protein, located before the start of the first beta-strand, and the last 24 residues, located past the end of the last beta-strand, are relatively flexible; this contrasts with the relatively rigid and well-defined structure of the beta-sheet. Amino acid sequence comparisons among a diverse set of species indicate that the A. fulgidus protein is homologous to the human RNase P protein Rpp29, yeast RNase P protein Pop4, and a known archaeal RNase P protein from Methanobacter thermoautotrophicus; conserved hydrophobic residues indicate that the homologous protein in each of these species contains a similar beta-sheet structure. Conserved surface residues located in the loop connecting strands beta2 and beta3, the loop connecting strands beta4 and beta5, and in the flexible N- and C-terminal tails are most likely to have specific interactions with the RNA and other proteins of RNase P. The structural model of an RNase P protein component provided by the present work provides an essential step toward eventually understanding the overall architecture of this complex enzyme and the mechanism by which it performs its functions.