Thiamin biosynthesis: still yielding fascinating biological chemistry.

The present paper describes the biosynthesis of the thiamin thiazole in Bacillus subtilis and Saccharomyces cerevisiae. The two pathways are quite different: in B. subtilis, the thiazole is formed by an oxidative condensation of glycine, deoxy-D-xylulose 5-phosphate and a protein thiocarboxylate, whereas, in S. cerevisiae, the thiazole is assembled from glycine, NAD and Cys205 of the thiazole synthase.

Stepwise evolution of protein native structure with electrospray into the gas phase, 10(-12) to 10(2) s.

Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently "gentle" to introduce nonvolatile biomolecules such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although in some cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the solution structure of large protein complexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations. The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as proposed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures. Control of individual experimental factors allows optimization for specific research objectives.

Multi-step evolution of protein conformation on electrospray into the gas phase.

In the gas phase, some properties of native versus denatured protein conformations correspond to those in solution, such as affinity for protons and physical cross section. However, the capacity for hydrogen/deutrerium exchange is the opposite, with ubiquitin 7+ and 13+ ions exchanging >-60 D and approximately 15 D atoms, respectively. A variety of experimental methods now delineate a series of conformational perturbations that can occur in the 10(-12) s to 10(+2) s following electrospray, including side-chain collapse, hydrophobic and electrostatic non-covalent bond unfolding and refolding into a variety of non-native structures.

IgE receptor-mediated alteration of membrane-cytoskeleton interactions revealed by mass spectrometric analysis of detergent-resistant membranes.

We use electrospray ionization mass spectrometry to quantify >100 phospholipid (PL) components in detergent-resistant membrane (DRM) domains that are related to ordered membrane compartments commonly known as lipid rafts. We previously compared PL compositions of DRMs with plasma membrane vesicles and whole cell lipid extracts from RBL mast cells, and we made the initial observation that antigen stimulation of IgE receptors (FcepsilonRI) causes a significant change in the PL composition of DRMs [Fridriksson, E. K., et al. (1999) Biochemistry 38, 8056-8063]. We now characterize the signaling requirements and time course for this change, which is manifested as an increase in the recovery of polyunsaturated PL in DRM, particularly in phosphatidylinositol species. We find that this change is largely independent of tyrosine phosphorylation, stimulated by engagement of FcepsilonRI, and can be activated by Ca(2+) ionophore in a manner independent of antigen stimulation. Unexpectedly, we found that inhibitors of actin polymerization (cytochalasin D and latrunculin A) cause a similar, but more rapid, change in the PL composition of DRMs in the absence of FcepsilonRI activation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane domains. Consistent with this interpretation, a membrane-permeable stabilizer of F-actin, jasplakinolide, prevents antigen-stimulated changes in DRM PL composition. These results are confirmed by a detailed analysis of multiple experiments, showing that receptor and cytochalasin D-stimulated changes in DRM lipid composition follow first-order kinetics. Analysis in terms of the number of double bonds in the fatty acid chains is valid for total PL of the major headgroups and for headgroups individually. In this manner, we show that, on average, concentrations of saturated or monounsaturated PL decrease in the DRM, whereas concentrations of PL with two or more double bonds (polyunsaturated PL) increase due to cytoskeletal perturbation. We find that these changes are independent of fatty acid chain length. Our mass spectrometric analyses provide a detailed accounting of receptor-activated alterations in the plasma membrane that are regulated by the actin cytoskeleton.

Early structural evolution of native cytochrome c after solvent removal.

Electrospray ionization transfers thermally labile biomolecules, such as proteins, from solution into the gas phase, where they can be studied by mass spectrometry. Covalent bonds are generally preserved during and after the phase transition, but it is less clear to what extent noncovalent interactions are affected by the new gaseous environment. Here, we present atomic-level computational data on the structural rearrangement of native cytochrome c immediately after solvent removal. The first structural changes after desolvation occur surprisingly early, on a timescale of picoseconds. For the time segment of up to 4.2 ns investigated here, we observed no significant breaking of native noncovalent bonds; instead, we found formation of new noncovalent bonds. This generally involves charged residues on the protein surface, resulting in transiently stabilized intermediate structures with a global fold that is essentially the same as that in solution. Comparison with data from native electron capture dissociation experiments corroborates both its mechanistic postulations and our computational predictions, and suggests that global structural changes take place on a millisecond timescale not covered by our simulations.

Top-down identification and characterization of biomolecules by mass spectrometry.

The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10(5)) mass resolving power and accuracy ( approximately 1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and post-translational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation." The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.

Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics.

For the characterization of protein sequences and post-translational modifications by MS, the 'top-down' proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used 'bottom-up' approach, which instead uses such data of peptides from the protein's digestion, but the top-down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False-positive rates for the identification of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient. Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top-down approach has a approximately 500 residue, approximately 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas-phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray ('prefolding dissociation'). This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues.

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.

Simultaneous kinetic characterization of multiple protein forms by top down mass spectrometry.

Top down mass spectrometry, using a Fourier transform instrument, has unique capabilities for biomolecule kinetic studies, in that the concentration of large molecules in a reaction mixture can be monitored simultaneously from its mass spectrum produced by electrospray ionization. This is demonstrated with enzyme modifications occurring in the biosynthesis of the thiazole moiety of thiamin phosphate. The formation rate of ThiS-thiocarboxylate from ThiS was determined from the relative abundance of the corresponding m/z 10162 and 10146 isotopic peak clusters for all the observable charge states in the mass spectra measured at different reaction times. Even without measuring standard ionization efficiencies, the rate and precision of 0.018 +/- 0.004 min(-1) agree well with the 0.027 +/- 0.003 min(-1) obtained with a radiochemical assay, which requires a separate derivatization step. To illustrate the simultaneous characterization of the reaction kinetics of a native enzyme and its mutant, the imine formation rate of ThiG and its substrate DXP was compared between the native protein (M(r) = 26803.9) and its E98A (M(r) = 26745.9) or D182A (M(r) = 26759.9) mutant in the same reaction mixture. The kinetic data show clearly that neither the E98 nor the D182 residues participate in the imine formation. The high resolution and MS/MS capabilities of FTMS should make possible the extension of this kinetics approach to far more complicated systems, such as simultaneous monitoring of 24 native, intermediate, and reduced forms in the reductive unfolding of a mixture of ribonuclease A and the five isoforms of ribonuclease B. Stable intermediates with different SS bonding (same molecular weight) can be differentiated by MS/MS, while molecular ions differing by only 2 Da are distinguished clearly by synthesizing isotopically depleted proteins.

The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS.

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.

Simultaneous characterization of the reductive unfolding pathways of RNase B isoforms by top-down mass spectrometry.

A novel method for characterization of the simultaneous reductive unfolding pathways of five isoforms of bovine pancreatic ribonuclease B (RNase B) is demonstrated. The results indicate that each isoform unfolds reductively through two three-disulfide-containing structured intermediates before proceeding to the fully reduced form, as in the reductive unfolding pathways of the A variant lacking the carbohydrate chain. The rates of reduction of bovine pancreatic ribonuclease A (RNase A) and RNase B and the formation and consumption of their reductive intermediates are identical, indicating that the unfolding events necessary to expose disulfide bonds for reduction are not affected by the oligosaccharide. The method utilizes top-down mass spectrometry and a naturally occurring tag on the protein, viz. the carbohydrate moiety, to obtain unfolding information of an ensemble of protein isoforms and is a generally applicable methodological advance for conducting folding studies on mixtures of different proteins.

Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry.

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 +/- 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R(28)-V(31), E(95)-F(107), and K(216) of 22%, 28%, and 34%, respectively, but with a possible (approximately 4%) fourth site at V(125)-D(150). However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable approximately 13% oxygen at the V(125)-D(150) site. For the "bottom-up" approach, detection of the approximately 4% oxidation at the V(125)-D(150) site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just approximately 4% of this, but the uniqueness of its mass value (approximately 1-2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V(125)-D(150) until K(216) is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K(216), H(137), and D(139) are conserved residues.

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry.

Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values. However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective.

Charge site mass spectra: conformation-sensitive components of the electron capture dissociation spectrum of a protein.

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c (m+) and z (m+•) products of cleavage at each of its molecular ion's inter-residue bonds. For example, the ECD spectra of ubiquitin (M + nH)(n+) ions, n = 7-13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ((1-7)+) and eight z ((1-8)+•) spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of "charge site (CS)" spectra, the c (m+) or z (m+•) products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

How ubiquitin unfolds after transfer into the gas phase.

The structural evolution of ubiquitin after transfer into the gas phase was studied by electron capture dissociation. Site-specific fragment yields show that ubiquitin's solution fold is overall unstable in the gas phase, but unfolding caused by loss of solvent is slowest in regions stabilized by salt bridges.