Formation Of Disulphide Bridges Research Articles

Post-translational modulation of cell signalling through protein succinylation

Cells need to adapt their activities to extra- and intracellular signalling cues. To translate a received extracellular signal, cells have specific receptors that transmit the signal to downstream proteins so that it can reach the nucleus to initiate or repress gene transcription. Post-translational modifications (PTMs) of proteins are reversible or irreversible chemical modifications that help to further modulate protein activity. The most commonly observed PTMs are the phosphorylation of serine, threonine, and tyrosine residues, followed by acetylation, glycosylation, and amidation. In addition to PTMs that involve the modification of a certain amino acid (phosphorylation, hydrophobic groups for membrane localisation, or chemical groups like acylation), or the conjugation of peptides (SUMOylation, NEDDylation), structural changes such as the formation of disulphide bridge, protein cleavage or splicing can also be classified as PTMs. Recently, it was discovered that metabolites from the tricarboxylic acid (TCA) cycle are not only intermediates that support cellular metabolism but can also modify lysine residues. This has been shown for acetate, succinate, and lactate, among others. Due to the importance of mitochondria for the overall fitness of organisms, the regulatory function of such PTMs is critical for protection from aging, neurodegeneration, or cardiovascular disease. Cancer cells and activated immune cells display a phenotype of accelerated metabolic activity known as the Warburg effect. This metabolic state is characterised by enhanced glycolysis, the use of the pentose phosphate pathway as well as a disruption of the TCA cycle, ultimately causing the accumulation of metabolites like citrate, succinate, and malate. Succinate can then serve as a signalling molecule by directly interacting with proteins, by binding to its G protein-coupled receptor 91 (GPR91) and by post-translationally modifying proteins through succinylation of lysine residues, respectively. This review is focus on the process of protein succinylation and its importance in health and disease.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Physiological role of reactive oxygen species in testis and epididymal spermatozoa.

The reactive oxygen species (ROS) play an important role in various aspects of male reproductive function, for spermatozoa to acquire the ability to fertilize. However, the increase in ROS generation, both due to internal and external factors, can induce oxidative stress, causing alterations in the structure and function of phospholipids and proteins. In the nucleus, ROS attack DNA, causing its fragmentation and activation of apoptosis, thus altering gene and protein expression. Accumulating evidence also reveals that endogenously produced ROS can act as second messengers in regulating cell signalling pathways and in the transduction of signals that are responsible for regulating spermatogonia self-renewal and proliferation. In the epididymis, they actively participate in the formation of disulphide bridges required for the final condensation of chromatin, as well as in the phosphorylation and dephosphorylation of proteins contained in the fibrous sheath of the flagellum, stimulating the activation of progressive motility in epididymal spermatozoa. In this review, the role of small amounts of ROS during spermatogenesis and epididymal sperm maturation was discussed.

Clearance of oxytocin and its potentially enzyme resistant analogues in the OXT-receptor compartment of the potassium depolarized rat myometrium.

The time–response behaviour of a group of oxytocin analogues structurally modified on potential sites of oxytocin splitting by tissue inactivation enzymes (“enzyme probes”) was investigated ex vivo on the potassium depolarized rat myometrium (at 30°C) and compared with the data obtained in the in vivo experiments. The modified oil‐immersion method by Kalsner and Nickerson was used to record time profiles after cessation of a steady state myometrium contraction triggered by analogues in a high potassium tissue medium. An exchange of the aqueous medium for mineral oil enables to suppress return diffusion of the peptide and to record its irreversible clearance near the corresponding receptor compartment. Response records were analysed by a nonlinear numeric procedure based on combination of steady state and kinetic terms that allows concomitant estimations of affinities from time–response measurements, in the given case for analogues on depolarized myometrium. Potential inactivation‐sensitive sites in the oxytocin chain are the Ν‐terminal peptide bond Cys1‐Tyr2 (aminopeptidase splitting), the intramolecular disulphide bridge (reduction and formation of the practically inactive linear peptide) and the C‐terminal Leu8‐GlyNH2 9 or the Pro7‐Leu8 (postprolin cleaving enzyme) bond, respectively. Clearance rate constants of single peptides in the OXT‐receptor compartment were in an interval of 0.025 to 0.28 min−1. The fragment contribution analysis reveals a significant linear additivity of individual structural changes and thus a predictivity of irreversible inactivation rate in the receptor compartment. The most potent inactivation of oxytocin is associated with aminopeptidase splitting; other enzymes may play some though nondecisive role. Less significant differences within the peptide group were found for rate constants for peptide transport between receptor compartment and its external aqueous medium. Besides rate constants, the evaluation of time–response data yields affinity values of the tested peptides and indicates a 25‐times desensitation of depolarized compared with a native state.

Membrane interaction and disulphide-bridge formation in the unconventional secretion of Tau.

Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer’s disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.

Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRSapm)

In crystal structure, the homodimeric (α2) mimi-virus Tyrosyl-tRNA Synthetases (TyrRSapm) showed significant variation in dimer orientation as compared to other solved structures of TyrRSs. Previous report showed that Bacillus stearothermophilus N-terminal TyrRS exists as dimer under native condition and unfolds through a monomeric intermediate. In our previous studies, we have reported that the C-terminal anticodon binding domain of TyrRSapm might have an indirect role in noncanonical dimer formation. To study the implication of the non-canonical dimer interface on the structural and functional organization of TyrRSapm, we replaced all three W residues (both of dimer interface and C-terminal domain) systematically with F residues (using site directed mutagenesis) with four different combinations (W80F/W120F, W278, W80F/W278F, W120F/W278F). We took advantage of Trp (W) fluorescence as the biophysical reporter for probing dimer interface study. The circular dichroism (CD) spectra of Trp mutants are dramatically different from wild-type indicating a huge conformation reorientation due to mutations. Another unusual feature of this enzyme is that it contains 10 cysteine residues per monomer and none of them involved in disulphide bridge formation. Using DTNB reaction as a probe again it was observed that mutants behave differently indicating a major perturbation. Fluorescence anisotropy supported by analytical ultracentrifugation data showed that TyrRSapm and its mutants exist as dimer. The unfolding pattern of N-terminal and C-terminal domain shows significant difference from each other indicating differential melting of domains. Aminoacylation reaction showed that two of the four Trp. mutants retain enzyme activities though different as compared to WT, while the other two mutants lost their activities. Hence it can be concluded that, the Trp residues play a crucial role in the structural as well as functional organization of TyrRSapm that may be reflected as the non canonical orientation of dimer conformation in crystal structure.

Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).

Unravelling of the water-binding capacity of cold-gelated whey protein microparticles

Whey protein microparticles (CG MPs) were made with a cold gelation method. Without shearing or mixing during gelation spherical CG MPs were formed, while shearing or mixing resulted in smaller irregularly shaped CG MPs. The water-binding capacity of pellets (WBC-P) that were obtained after centrifuging CG MP dispersions was remarkably large (11–18 g water/g dry matter), though this value decreased at larger centrifugation speeds.Microscopy images hinted at the presence of two water domains in the CG MP pellets: water within and between the CG MPs. The images also imply that the amount of water within the CG MPs was determined by the centrifugation speed. The amount of water between CG MPs seemed to be defined by the amount of particle-particle interactions that were present, as suggested by the effects of the particles' size and the inhibition of the disulphide bridge formation on the WBC-P.Although microscopy images showed two water domains, only one main peak was found with time domain nuclear magnetic resonance. This was explained by water diffusion from one water domain to the other within the measuring time. This fast diffusion implies that the CG MPs had a relatively open structure. Overall, it was concluded that water-binding by CG MPs was affected by various factors and that a good understanding of the water-binding requires the use of a range of measurements.

HLA-B*38:55Q: a new alternatively expressed allele identified in a three-generation Italian family.

The new allelic variant HLA-B*38:55Q differs from the closest related B*38:01:01 by one nucleotide substitution at position 373 in exon 3 (TGC>CGC). This results in a difference of one amino acid at residue 101 of the HLA-B heavy chain, from a neutral-polar Cys to a basic-polar Arg, thus impairing disulphide bridge formation in the alpha-2 domain. This alteration of the secondary structure probably affects the maturation of the heavy chain and the level of surface expression, making the HLA-B*38:55Q undetectable by standard serological typing.

Immunohistochemical expression of sulfhydryl oxidase (QSOX1) in pediatric medulloblastomas.

BackgroundMedulloblastoma is a malignant, invasive embryonal tumor of the cerebellum and accounts for 20% of intracranial tumors in children. QSOX1, whose functions include formation of disulphide bridges, which are needed for correct protein folding and stability, formation of the extracellular matrix, regulation of the redox status and cell cycle control, appears to be involved in apoptosis in pathological states such as cancer. Thus, the aim of this study was to investigate the immunohistochemical expression of QSOX1 in medulloblastomas and nonneoplastic cerebellum.MethodsHistology blocks of pediatric medulloblastomas were separated and two representative areas of the tumors and non-neoplastic cerebellum samples were used to construct tissue microarrays (TMAs) that were stained with an anti-QSOX1 antibody, and the slides were read using image analysis software.ResultsQSOX1 immunoexpression was observed in the non-neoplastic cerebellum samples and the medulloblastoma samples. There was no statistically significant relationship between QSOX1 immunopositivity in the medulloblastoma samples and the clinical and pathological variables.ConclusionsAlthough QSOX1 did not prove useful for stratifying patients into risk groups, tumor cells and the fibrillar extracellular matrix were positive for this marker, indicating that this enzyme may be involved in the pathogenesis of medulloblastoma.Virtual SlidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1822040654139436

Modulating the structure of EGFR with UV light: new possibilities in cancer therapy.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.

Understanding the differences in gelling properties between lupin protein isolate and soy protein isolate

The gelling properties of lupin protein isolate (LPI) were compared with those of soy protein isolate (SPI). It was found that LPI behaves fundamentally different than SPI, evidenced by the formation of weaker and deformable gels. Further investigation shows that both protein isolates can be considered particle gels and that LPI particles do not swell as much as SPI particles inside the network. Besides, heating hardly affects LPI particles while SPI particles show additional swelling. To explain the differences, the sulfhydryl reactivity of LPI was tested. The amount of free sulfhydryl groups on LPI was higher than the amount of free sulfhydryl groups on SPI. Upon heating the amount of free sulfhydryl groups on LPI increases. We hypothesize that the compact, heat stable structure of the protein particles suppresses the intermolecular bonding through disulphide bridge formation and favours intramolecular crosslinking. The small sulphur-rich proteins that are not incorporated within the particles but are present in the surrounding solution cannot strengthen the particle network, due to their low concentration. LPI did not form gels of similar consistency as SPI and may therefore be less useful for solid food products. The thermal stability of LPI could offer opportunities for high-protein foods that require low viscosity after heating.

Secretion and functional display of fusion proteins through the curli biogenesis pathway

Curli are functional amyloids expressed as fibres on the surface of Enterobacteriaceae. Contrary to the protein misfolding events associated with pathogenic amyloidosis, curli are the result of a dedicated biosynthetic pathway. A specialized transporter in the outer membrane, CsgG, operates in conjunction with the two accessory proteins CsgE and CsgF to secrete curlin subunits to the extracellular surface, where they nucleate into cross-beta strand fibres. Here we investigate the substrate tolerance of the CsgG transporter and the capability of heterologous sequences to be built into curli fibres. Non-native polypeptides ranging up to at least 260 residues were exported when fused to the curli subunit CsgA. Secretion efficiency depended on the folding properties of the passenger sequences, with substrates exceeding an approximately 2 nm transverse diameter blocking passage through the transport channel. Secretion of smaller passengers was compatible with prior DsbA-mediated disulphide bridge formation in the fusion partner, indicating that CsgG is capable of translocating non-linear polypeptide stretches. Using fusions we further demonstrate the exported or secreted heterologous passenger proteins can attain their native, active fold, establishing curli biogenesis pathway as a platform for the secretion and surface display of small heterologous proteins.

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation.

The neuropeptide transcriptome of a model echinoderm, the sea urchin Strongylocentrotus purpuratus

Neuronal secretion of peptide signaling molecules (neuropeptides) is an evolutionarily ancient feature of nervous systems. Here we report the identification of 20 cDNAs encoding putative neuropeptide precursors in the sea urchin Strongylocentrotus purpuratus (Phylum Echinodermata), providing new insights on the evolution and diversity of neuropeptides. Identification of a gonadotropin-releasing hormone-like peptide precursor (SpGnRHP) is consistent with the widespread phylogenetic distribution of GnRH-type neuropeptides in the bilateria. A protein (SpTRHLP) comprising multiple copies of peptides that share structural similarity with thyrotropin-releasing hormone (TRH) is the first TRH-like precursor to be identified in an invertebrate. SpCTLP is the first calcitonin-like peptide with two N-terminally located cysteine residues to be found in a non-chordate species. Discovery of two proteins (SpPPLNP1, SpPPLNP2) comprising homologs of molluscan pedal peptides and arthropod orcokinins indicates the existence of a bilaterian family of pedal peptide/orcokinin-type neuropeptides. Other proteins identified contain peptides that do not share apparent sequence similarity with known neuropeptides. These include Spnp5, which comprises multiple copies of C-terminally amidated peptides that have an N-terminal Ala-Asn motif (AN peptides), and Spnp9, Spnp10 and Spnp12, which contain putative neuropeptides with a C-terminal Phe-amide, Ser-amide or Pro-amide, respectively. Several proteins (Spnp11, 14, 15, 16, 17, 18, 19 and 20) contain putative neuropeptides with multiple cysteine residues (2, 6 or 8), which may mediate formation of intramolecular or intermolecular disulphide bridges. Looking ahead, the identification of these neuropeptide precursors in S. purpuratus has provided a strong basis for a comprehensive analysis of neuropeptide function in this model echinoderm species.

The effect of methionine to cysteine substitution on the stability of formate dehydrogenase from Candida methylica

Abstract Because of its industrial importance, the FDH enzyme has been subjected to several protein engineering studies in order to stabilise it at high temperatures. In this study, methionine to cysteine substitution (M1C) and disulphide bridge formation between residues 1 (M1C) and 62 (D62C) were investigated in terms of their effect on the thermostability of cm FDH. Although the mutant cm FDHs, D62C and M1C/D62C, did not show the improvement expected in its stability, characterisation studies of individual mutants showed that the M1 residue is related to temperature stability. Catalytic efficiency ( k cat / K M ) of the M1C single mutant was 63% better than that of the native cm FDH and the T m value of this mutant was measured as significantly higher than that of the native cm FDH by using fluorescence measurements and Circular Dichroism Spectra. Comparing to other studies related to Cys or Met substitution in the literature, 2 °C enhancement of T m is a promising result for the industrially important FDH enzyme and to understand the relationship between thermostability and Met to Cys substitution.

Molecular characterization and increased expression of the Nile tilapia, Oreochromis niloticus (L.), T‐cell receptor beta chain in response to Streptococcus agalactiae infection

The complete cDNA sequence of the Nile tilapia T-cell receptor (TCR) β chain was cloned using 5' RACE. The full-length, 1263-bp cDNA contained a 942-bp open reading frame (ORF) encoding a 314-amino-acid protein. Sequence analyses revealed that the Nile tilapia TCR β chain contains four conserved cysteine residues involved in the formation of disulphide bridges and a conserved amino acid motif believed to be important for assembly and signalling of the TCR αβ/CD3 complex, both of which are normally found in the TCR β chain of other vertebrates. As detected using semi-quantitative and quantitative RT-PCR, the highest expression level of TCR β was detected in the thymus. Interestingly, Streptococcus agalactiae significantly induced the up-regulation of the TCR β chain, and the strongest up-regulation was detected in the brain and peripheral blood leucocytes (PBLs). In in vitro experiments, concanavalin A and Aeromonas hydrophila were found to significantly increase the expression of the TCR β chain in PBLs after 48 h (P < 0.01) and 72 h (P < 0.05), respectively. Furthermore, real-time PCR analysis showed that intraperitoneal injection (IP) of 10(7) cfu mL(-1) of S. agalactiae could induce TCR β expression that was greater than the expression observed following administration of 10(9) cfu mL(-1). The presence of the TCR β chain in fish detected in this study suggests the presence of T-cell populations that have been found in higher vertebrates, which may play a crucial functional role in the response to fish pathogens.

Biophysical characterisation of the nucleocapsid protein from a highly pathogenic porcine reproductive and respiratory syndrome virus strain

The arterivirus nucleocapsid (N) protein is a multifunctional protein that binds viral RNA for encapsidation and has potential roles in host cell processes. This study characterised the N protein from a highly virulent North American strain of porcine reproductive and respiratory syndrome virus (PRRSV). The association with viral RNA was mapped to defined motifs on the N protein. The results indicated that disulphide bridge formation played a key role in RNA binding, offering an explanation why infectious virus cannot be rescued if cysteine residues are mutated, and that multiple sites may promote RNA binding.

Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

BackgroundThe Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.Methodology/Principal FindingsTransient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.Conclusions/SignificanceWe show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.

Use of Raman spectroscopy for the identification of radical-mediated damages in human serum albumin

Damages induced by free radicals on human serum albumin (HSA), the most prominent protein in plasma, were investigated by Raman spectroscopy. HSA underwent oxidative and reductive radical stress. Gamma-irradiation was used to simulate the endogenous formation of reactive radical species such as hydrogen atoms ((•)H), solvated electrons (e(aq)(-)) and hydroxyl radicals ((•)OH). Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and specific damages occurring at sensitive amino acid sites. In particular, the analysis of the S-S stretching region suggested the radical species caused modifications in the 17 disulphide bridges of HSA. The concomitant action of e(aq)(-) and (•)H atoms caused the formation of cyclic disulphide bridges, showing how cystine pairs act as efficient interceptors of reducing species, by direct scavenging and electron transfer reactions within the protein. This conclusion was further confirmed by the modifications visible in the Raman bands due to Phe and Tyr residues. As regards to protein folding, both oxidative and reductive radical stresses were able to cause a loss in α-helix content, although the latter remains the most abundant secondary structure component. β-turns motifs significantly increased as a consequence of the synergic action of e(aq)(-) and (•)H atoms, whereas a larger increase in the β-sheet content was found following the exposure to (•)OH and/or (•)H attack.