A small-molecule catalyst of protein folding in vitro and in vivo

BACKGROUND: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo'). RESULTS: A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo. CONCLUSIONS: A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER.     

Determination of the DeltapKa between the active site cysteines of thioredoxin and DsbA

Thioredoxin superfamily members share a considerable degree of structural similarity, with a conserved CX(i)X(j)C motif at the active site, where C stand for two cysteines that alternate between a reduced thiol and oxidized disulfide states, and X(i)and X(j) are two amino acids different in each family member. Despite these similarities, they display very different redox potentials and pKas for the active site dithiol, and fulfill different physiological roles. Thioredoxin, for example, promotes the reduction of disulfide bonds, while DsbA promotes their oxidation in prokaryotic cells. The factors that promote these differences are still not fully understood. However, it is generally accepted that the different stabilities of the redox active disulfide bond depends on the degree of stabilization, in the reduced state, of the thiolate of one of the active site cysteines (nucleophilic cysteine). In this work we have used QM/MM methods to compare and characterize the active site dithiols of both enzymes, and to shed some light on the structural features responsible for the large differences in pKa and redox potential between two homologous enzymes, thioredoxin and DsbA. We have also analyzed the main factors pointed out in the literature as responsible for their different properties. We obtained the value of 4.5 for pKa difference (DeltapKa) between the nucleophilic cysteines of both enzymes, which is in excellent agreement with most of the experimental values. Additionally, we found that the principal differentiating factor responsible for this observed DeltapKa are the alpha2-alpha helices, which greatly contribute to the mentioned value, by stabilizing the DsbA thiolate in a much greater extend than the thioredoxin thiolate. A double mutation of the conserved residues Asp26 and Lys57, in thioredoxin, and Glu24 Lys58, in DsbA, by alanines did not change the DeltapKa value; this supports the hypothesis that these residues are not involved in the differentiation of the properties of the active centre dithiol. However, we found out that these residues are important for the stabilization of the nucleophilic thiolate. The X(i) and X(j) residues also do not seem to promote the stabilization of the thiolates. In fact, the corresponding double alanine mutants are more stable than the wild-type enzymes. However, these residues are involved in the differentiation between thioredoxin and DsbA, stabilizing the DsbA thiolate by a larger extent than the thioredoxin thiolate.     

Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and M?ssbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.     

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.     

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2–4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide–acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and peptide–acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation. © 1998 John Wiley & Sons, Ltd.     

Structural and functional diversity of glutaredoxins in yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.     

Structure, stability, and orientation of BSA adsorbed to silica

In this investigation, the structure, stability, and orientation of bovine serum albumin (BSA) adsorbed onto silica particles were studied using differential scanning calorimetry (DSC) and limited proteolysis in combination with mass spectrometry (MS). DSC gave information on the overall structural stability of BSA while limited proteolysis was used to probe the accessibility of enzymatic cleavage sites, thereby yielding information on the orientation and structure of BSA adsorbed to silica surfaces. Thermal investigation of BSA in various buffers, both free in solution and in the adsorbed state, showed that solutes that surround the protein played an important role with respect to the overall structural stability and the structural heterogeneity of BSA. Limited proteolysis with trypsin and chymotrypsin indicated that BSA in the adsorbed state is oriented with domain 2 facing the silica surface. Also, upon adsorption, no additional cleavage sites were exposed. The combination of the results presented in this study implied that BSA molecules adsorbed onto silica particles were significantly reduced in their structural stability, but not to an extent that internal residues within the native structure became fully exposed to the solution.     

Characterization of bovine surfactant proteins B and C by electrospray ionization mass spectrometry

Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.     

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.Abbreviations 2D Two-dimensional - CE Capillary electrophoresis - CID Collision-induced dissociation - ECD Electron capture dissociation - ESI Electrospray ionisation - FT-ICR Fourier transform ion cyclotron resonance - HPLC High performance liquid chromatography - ICAT Isotope coded affinity tags - ICP Inductively-coupled plasma - IDA Immino-diacetic acid - IMAC Immobilised metal affinity chromatography - IRMPD Infrared multiphoton dissociation - IT Ion trap - MALDI Matrix-assisted laser desorption/ionisation - MRP14 Myeloid-related protein 14 - MS Mass spectrometry - NTA Nitrilo-triacetic acid - PAGE Polyacrylamide gel electrophoresis - PDI Protein disulfide isomerase - pS Phosphoserine residue - PSD Post-source decay - pT Phosphothreonine residue - PVDF Polyvinylidene fluoride - pY Phosphotyrosine residue - Q-TOF Quadrupole-time-of-flight - RP Reversed phase - SIM Single-ion monitoring - SDS Sodium dodecyl sulfate - SORI Sustained off-resonance irradiation - TLC Thin-layer chromatography - TOF Time-of-flight An erratum to this article can be found at     

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.     

Synthesis, redox properties, and conformational analysis of vicinal disulfide ring mimics

A vicinal disulfide ring (VDR) results from disulfide-bond formation between two adjacent cysteine residues. This eight-membered ring is a rare motif in protein structures and is functionally important to those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR mimics, discernment of the preferred conformation of these mimics, and the determination of their respective disulfide redox potentials.     

Proton- and redox-controlled switching of photo- and electrochemiluminescence in thiophenyl-substituted boron-dipyrromethene dyes

A luminescent molecular switch in which the active thiol/disulfide switching element is attached to a meso-phenyl-substituted boron-dipyrromethene (BDP) chromophore as the signalling unit is presented. The combination of these two functional units offers great versatility for multimodal switching of luminescence: 1) deprotonation/protonation of the thiol/thiolate moiety allows the highly fluorescent meso-p-thiophenol-BDP and its nonfluorescent thiolate analogue to be chemically and reversibly interconverted, 2) electrochemical oxidation of the monomeric dyes yields the fluorescent disulfide-bridged bichromophoric dimer, also in a fully reversible process, and 3) besides conventional photoexcitation, the well separated redox potentials of the BDP also allow the excited BDP state to be generated electrochemically (i.e., processes 1) and 2) can be employed to control both photo- and electrochemiluminescence (ECL) of the BDP). The paper introduces and characterizes the various states of the switch and discusses the underlying mechanisms. Investigation of the ortho analogue of the dimer provided insight into potential chromophore-chromophore interactions in such bichromophoric architectures in both the ground and the excited state. Comparison of the optical and redox properties of the two disulfide dimers further revealed structural requirements both for redox switches and for ECL-active molecular ensembles. By employing thiol/disulfide switching chemistry and BDP luminescence features, it was possible to create a prototype molecular ensemble that shows both fully reversible proton- and redox-gated electrochemiluminescence.     

Quantitative assessment of cysteine and cystine in peptides and proteins following organomercurial derivatization and analysis by matrix-assisted laser desorption ionization mass spectrometry

Protocols for the analysis of the sulfhydryl content in peptides and proteins using chemical derivatization by organomercurial reagents and analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have been developed. The number of reactive cysteine residues in peptides and proteins can be determined by exploiting the affinity and selectivity of organomercurial reagents for macromolecular thiols. Mass shifts observed in MALDI mass spectra obtained before and after cysteine derivatization with p-hydroxy-mercuribenzoate (pHMB) permit the number of free sulfhydryl groups to be determined. The pHMB derivative of each free cysteine residue provides a mass shift of 321 u, overcoming limitations in the mass resolution of MALDI time-of-flight mass spectrometry. Reactive cysteine residues in a macromolecule can be selectively derivatized by using a fivefold molar excess of pHMB reagent. Total sulfhydryl content (i.e., cysteine and cystine) can be determined after disulfide reduction. However, analyses for total cysteine content are more complex, requiring protein denaturation, cystine reduction, and sample purification before derivatization and analysis by MALDI-MS. Conditions for sample denaturation, alkyl-phosphine reduction, pHMB derivatization, and sample purification by analyte adsorption and desalting on protein transfer membranes, are described for cysteine/cystine analysis performed on microgram (10–200 pmol) quantities of somatostatin, insulin, hemoglobin, and β-lactoglobulin.     

一种新型独特芋螺毒素的分离与结构鉴定

从中国南海的一种食虫芋螺独特芋螺(Conus caracteristicus)的毒液中分离得到一种新的芋螺毒素, 对其进行了结构鉴定和化学合成. 结果表明, 这是一种新的T-超家族芋螺毒素.     

Mass spectrometry profiles superoxide-induced intramolecular disulfide in the FMN-binding subunit of mitochondrial complex I

Protein thiols with regulatory functions play a critical role in maintaining the homeostasis of the redox state in mitochondria. One major host of regulatory cysteines in mitochondria is Complex I, with the thiols primarily located on its 51 kDa FMN-binding subunit. In response to oxidative stress, these thiols are expected to form intramolecular disulfide bridges as one of their oxidative post-translational modifications. Here, to test this hypothesis and gain insights into the molecular pattern of disulfide in Complex I, the isolated bovine Complex I was prepared. Superoxide (O(2)(.-)) is generated by Complex I under the conditions of enzyme turnover. O(2)(.-)-induced intramolecular disulfide formation at the 51, kDa subunit was determined by tandem mass spectrometry and database searching, with the latter accomplished by adaptation of the in-house developed database search engine, MassMatrix [Xu, H., et al., J. Proteome Res. 2008, 7, 138-144]. LC/MS/MS analysis of tryptic/chymotryptic digests of the 51 kDa subunit from alkylated Complex I revealed that four specific cysteines (C(125), C(142), C(187), and C(206)) of the 51 kDa subunit were involved in the formation of mixed intramolecular disulfide linkages. In all, three cysteine pairs were observed: C(125)/C(142), C(187)/C(206), and C(142)/C(206). The formation of disulfide bond was subsequently inhibited by superoxide dismutase, indicating the involvement of O(2)(.-). These results elucidated by mass spectrometry indicate that the residues of C(125), C(142), C(187), and C(206) are the specific regulatory cysteines of Complex I and they participate in the oxidative modification with disulfide formation under the physiological or pathophysiological conditions of oxidative stress.     

Spectroscopic evidence for site specific chemistry at a unique iron site of the [4Fe-4S] cluster in ferredoxin:thioredoxin reductase

Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe-4S] in close proximity to a redox active disulfide. M?ssbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe-4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the N-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe-4S]2+ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe-4S]3+ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe-S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe-S enzymes.