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BBA - Proteins and Proteomics (v.1814, #3)
A combination method of chemical with enzyme reactions for identification of membrane proteins by Jeong-Eun Lee; Joseph Kwon; Moon-Chang Baek (pp. 397-404).
A simple method for effective analysis of various proteins has been developed, including membrane proteins, with LC–MS/MS, using CNBr and acetic acid cleavage in one reaction for the digestion of both the M/ and /D/ positions within the target proteins. This dual chemical reaction has been compared with traditional CNBr or an acid cleavage method using a rat kidney membrane fraction and it showed an advantage of the dual reaction with respect to a high number of peptides detected and a high protein recovery. Furthermore, when this dual chemical reaction was combined with trypsin digestion, the number of proteins surprisingly increased approximately 3.0 times more than in the cases with the trypsin digestion only. It was also 1.9 times more than in cases dealing with Tube-Gel trypsin digestion, which is one of the most efficient digestion methods. In addition, it was shown that this dual chemical reaction could be applied to an in-gel digestion. Using the combination of the chemical and enzyme reaction, 172 proteins including 95 membrane proteins were identified. This indicated that this method is one of the efficient systems in single MS/MS analysis. In particular, many membrane proteins identified in this study were detected by a new combination, but not by a traditional trypsin digestion method.Display Omitted► CNBr and acetic acid cleave N-term of D as well as C-term of M and D residues. ► Chemicals have denaturing effect on membrane lipid bilayer structure. ► Dual reaction of chemicals-trypsin increases the membrane protein identification.
Keywords: Abbreviations; ABC; ammonium bicarbonate; ACN; acetonitrile; CNBr; cyanogen bromide; FDR; false discovery rate; LC–MS/MS; liquid chromatography tandem mass spectrometry; TMDs; transmembrane domainsChemical cleavage; Membrane protein; Kidney membrane; LC–MS/MS
Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms by Changjian Feng; Weihong Fan; Dipak K. Ghosh; Gordon Tollin (pp. 405-408).
The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H4B) cofactor andl-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition ofl-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.►The rates of bimolecular CO rebinding to the nNOS and iNOS oxygenase proteins were determined. ►L-Arg binding to murine iNOS N115L mutant gives much faster CO rebinding rate, compared to the wild type. ►Rat nNOS mutants at the equivalent site (L337N and L337F) have similar kinetics profile as the wild type. ►The distal isoform-specific residue N115 in murine iNOS may control heme ligand accessibility.
Keywords: Abbreviations; NO; nitric oxide; NOS; nitric oxide synthase; iNOS; inducible NOS; nNOS; neuronal NOS; NOS; oxy; the oxygenase domain of nitric oxide synthase; CaM; calmodulin; CO; carbon monoxide; Fe; II; ferrous heme species; Fe; II; CO; ferrous CO species; dRF; 5-deazariboflavin; dRFH; •; 5-deazariboflavin semiquinone; H; 4; B; 6R-5,6,7,8-tetrahydrobiopterin; Arg; l; -arginineNitric oxide synthase; Kinetics; Mutation; Mechanism; Ligand binding
A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum by Francesca Lavatelli; Francesca Brambilla; Veronica Valentini; Paola Rognoni; Simona Casarini; Dario Di Silvestre; Vittorio Perfetti; Giovanni Palladini; Gabriele Sarais; Pierluigi Mauri; Giampaolo Merlini (pp. 409-419).
An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98–2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266–2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms.►A novel immunoprecipitation method for purifying monoclonal serum FLC was developed. ►Biochemical features of a pilot set of serum FLC were studied by proteomics. ►Monoclonal serum FLC were an heterogeneous populations of isoforms. ►The analyzed serum amyloidogenic FLC possessed post-translational modifications.
Keywords: Abbreviations; FLC; free light chains; LC; light chains; IEF; isoelectrofocusing; 2D-PAGE; two-dimensional polyacrylamide gel electrophoresis; MS; mass spectrometry; MALDI-TOF; matrix assisted laser desorption/ionization time of flight; PMF; peptide mass fingerprinting; DTT; dithiothreitol; LC-MS/MS; liquid chromatography tandem mass spectrometry; 2DC; two-dimensional chromatographyImmunoglobulin free light chains; Amyloidosis; Immunoprecipitation; Proteomics; Post-translational modifications
Inter-subunit interactions in erythroid and non-erythroid spectrins by Xiuli An; Xinhua Guo; Yang Yang; Walter B. Gratzer; Anthony J. Baines; Narla Mohandas (pp. 420-427).
Spectrins comprise α- and β-subunits made up predominantly of a series of homologous repeating units of about 106 amino acids; the α- and β-chains form antiparallel dimers by lateral association, and tetramers through head-to-head contacts between the dimers. Here we consider the first of these interactions. (1) We confirm earlier observations, showing that the first two paired repeats (βIR1 with αIR21, and βIR2 with αRI20) at one end of the erythroid spectrin (αIβI) dimer are necessary and sufficient to unite the chains; (2) we resolve a conflict in published reports by showing that the strength of the interaction is considerably increased on adding the adjoining pair of repeats (βIR3–αIR19); (3) in brain (αIIβII) spectrin the first two pairs of repeats are similarly essential and sufficient for heterodimer formation; (4) this interaction is~60-fold stronger than that in the erythroid counterpart, but no enhancement can be detected on addition of three further pairs of repeats; (5) formation of a tight αIβI dimer probably depends on structural coupling of the first two repeats in each chain; (6) an analysis of the sequences of the strongly interacting repeats, βIR1, βIIR1, αIR21 and αIIR20 and repeats in α-actinin, which also interact very strongly in forming an antiparallel dimer, affords a possible explanation for the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions, and also suggests the evolutionary path by which the erythroid and non-erythroid sequences diverged.► Similar to erythroid spectrin, the dimerization of brain spectrin also requires the first two pairs of repeats; ► the dimerization of brain spectrin is 60-fold stronger than that of erythroid counterpart; ► formation of αβ dimer for both erythroid and brain depends on structural coupling of the first two repeats in each chain; ► the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions could be attributed to the evolutionary path by which their sequences diverged.
Keywords: Abbreviations; CD; circular dichroism; GST; glutathione-S-transferase; His; histidine; SDS; sodium dodecylsulfate; IPTG; isopropyl β-; d; -thiogalactopyranoside; PAGE; polyacrylamide gel electrophoresis; PCR; polymerase chain reaction; SPR; surface plasmon resonanceSpectrin; Fodrin; Dimerization; Red cell
Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040 by Kazumi Funane; Yasuyuki Kawabata; Ryuichiro Suzuki; Young-Min Kim; Hee-Kwon Kang; Nobuhiro Suzuki; Zui Fujimoto; Atsuo Kimura; Mikihiko Kobayashi (pp. 428-434).
Cycloisomaltooligosaccharide glucanotransferase (CITase) belongs to glycoside hydrolase family 66. According to the sequence alignment of enzymes in the same family, we divided the structure of CITase into five regions from the N terminus to the C terminus: an N-terminal conserved region (Ser1–Gly403), an insertion region (R1; Tyr404–Tyr492), two conserved regions (R2; Glu493–Ser596 and R3; Gly597–Met700), and a C-terminal variable region (R4; Lys701–Ser934). CITase catalyzes the synthesis of cycloisomaltooligosaccharides (CIs) with 7–17 glucose units (CI-7 to CI-17) from dextran. In order to clarify the functions of these C-terminal regions (R1–R4), we constructed 15 deletion mutant enzymes. M123Δ (R4-deleted), MΔ234 (R1-deleted), and MΔ23Δ (R1/R4-deleted) catalyzed CI synthesis, but other mutants were inactive. M123Δ, MΔ234, and MΔ23Δ increased their Km values against dextran 40. The wild-type enzyme and M123Δ produced CI-8 predominantly, but MΔ234 and MΔ23Δ lost CI-8 production specificity. The kcat values of MΔ234 and MΔ23Δ decreased, and these mutants showed narrowed temperature and pH stability ranges. Our deletion analysis suggests that (i) R2 and R3 are crucial for CITase to generate an active form; (ii) both R1 and R4 contribute to substrate binding; and (iii) R1 also contributes to preference of CI-8 production and enzyme stability.Display Omitted► The C-terminal part of cycloisomaltooligosaccharide glucanotransferase (CITase) was divided into four regions, R1, R2, R3, and R4. R2 and R3 were crucial for forming enzymatically active CITase. R1 and R4 regions were shown to be carbohydrate-binding modules. R1 was demonstrated to be a structural element for substrate binding, control the size of producing cycloisomaltooctaose production (CI-8), and contribute to the stabilization of the enzyme.
Keywords: Abbreviations; CBM; carbohydrate-binding module; CD; cyclomaltodextrin; CGTase; CD glucanotransferase; CI; cycloisomaltooligosaccharide; CITase; CI glucanotransferase; GH; glycoside hydrolase family; HPLC; high-performance liquid chromatography; IG; isomaltooligosaccharide; PAGE; polyacrylamide gel electrophoresis; PCR; polymerase chain reaction; R1; R2, R3 and R4, regions of Tyr404–Tyr492, Glu493–Ser596, Gly597–Met700, and Lys701–Ser934 of CITase from; Bacillus circulans; strain T-3040, respectively; SDS; sodium dodecyl sulfate; TIM; triosephosphate isomerase Bacillus circulans; Cycloisomaltooligosaccharide; Cyclodextran; Cycloisomaltooligosaccharide glucanotransferase; C-terminal region
Osmoprotective proteome adjustments in mouse kidney papilla by B.J. Gabert; Kultz D. Kültz (pp. 435-448).
The papilla of the mammalian kidney must tolerate greatly varying degrees of hyperosmotic stress during urine concentration and depending on whole organism hydration state. To identify proteome adaptations supporting cell function and survival in such a harsh environment we compared the proteome of a) the hyperosmotic renal papilla with that of adjacent iso-osmotic cortex tissue and b) the renal papilla of diuretic versus that of anti-diuretic mice. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an iso-osmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were identified using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. We found 37 different proteins characteristic of the cortex and 16 proteins over-represented in the papilla. Regional specificity was confirmed by Western blot and further substantiated by immunohistochemistry for selected proteins. Proteins that are characteristic of the renal papilla include αB crystallin, Hsp beta-1, Hsp90, 14-3-3 protein, glutathione S-transferase, aldose reductase, actin and tropomyosin. Gene ontology analysis confirmed a significant increase in molecular functions associated with protein chaperoning and cell stabilization. Proteins over-represented in the cortex were largely related to routine metabolism. During antidiuresis 15 different proteins changed significantly while 18 different proteins changed significantly during diuresis relative to normally hydrated controls. Changes were confirmed by Western blot for selected proteins. Proteins that are significantly altered by diuretic state are associated with cell structure (actin, tubulin), signaling (Rho GDP dissociation inhibitor, abhydrolase domain-containing protein 14B), chaperone functioning (Hsp beta-1, αB crystallin, T complex protein-1) and anti-oxidant functions (α-enolase, GAPDH and LDH). Taken together our study reveals that specific proteins involved in protein folding, cytoskeletal stabilization, antioxidant responses, and stress signaling contribute greatly to the unique hyperosmotic stress resistant phenotype of the kidney papilla.Display Omitted► Heat shock proteins, anti-oxidant proteins, cytoskeletal proteins, and 14-3-3 are overrepresented in kidney medulla compared to cortex. ► Cytoskeletal proteins, molecular chaperones, and antioxidant proteins are also the main targets that are altered during antidiuresis in renal papilla. ► Therefore, hyperosmotic stress promotes a molecular phenotype that stabilizes proteins and the cytoskeleton in the renal papilla.
Role of the C -terminal domain of the regulatory subunit of AHAS isozyme III: Use of random mutagenesis with in vivo reconstitution (REM-ivrs) by Alexander Slutzker; Maria Vyazmensky; David M. Chipman; Ze'ev Barak (pp. 449-455).
In order to clarify the role of the C-terminal domain of the ilvH protein (the regulatory subunit of enterobacterial AHAS isozyme III, whose structure has been solved and reported by Kaplun et al., J Mol Biol357, 951, 2006) in the process of valine inhibition of AHAS III, we developed a procedure that randomly mutagenizes a specific segment of a gene through error-prone PCR and screens for mutants on the basis of the properties of the holoenzymes reconstituted in vivo (REM-ivrs). Previous work showed that the N-terminal domain includes the valine-binding ACT domain of the regulatory subunit and is sufficient to completely activate the catalytic subunit, but that this domain cannot confer valine sensitivity on the reconstituted enzyme. It appeared that the C-terminal domain of the ilvH is involved in some way in “signal transmission” of the inhibition by valine. As knowledge of the structure of AHAS holoenzymes and the interactions between the catalytic and regulatory subunits is very limited, a procedure that focuses on the C-terminal domain in the ilvH gene could add to the understanding of the mechanism by which the binding of valine to the regulatory subunit is coupled to inhibition of the catalytic activity. In the REM-ivrs procedure, a medium copy (~40 copies) plasmid expressing ilvH with a Valr mutation confers the Valr phenotype upon bacteria. All the single missense mutations produced by REM-ivrs were found to be localized to the interface between the C-terminal domains of two monomers in the ilvH dimer. The loss of specific contacts involved in inter-monomer interactions in this region might conceivably disrupt the structure of the C-terminal domain itself. Biochemical study of an isolated Valr mutant elicited by the REM-ivrs method detected no binding of radioactively labeled valine, as previously found in a truncation mutant. The idea that the C-terminal domain has a specific “signal-transmission” role was also contradicted by examination of the thermal stability of the Valr REM-ivrs variants by the Thermofluor method, which does not detect any signs of biphasic melting behavior for any of the mutants. We propose that the mutants of ilvH isolated by the REM-ivrs method differ from the wild-type in the equilibrium between two states of the enzyme. Without the specific interdomain contacts of the wild-type ilvH protein, the holoenzyme reconstituted from mutant regulatory subunits is apparently in a state with uninhibited activity and low affinity for valine.Display Omitted►Small subunit ilvH of heterooligomeric AHAS III required for valine-inhibition. ►ilvH C-terminal domain required for valine-inhibition but valine site in ACT domain. ►C-terminal domain mutants isolated by induction in-vitro + recomb-selection in-vivo. ►Random mutants mapped to region of interdomain contacts in the known structure. ►ilvH C-terminal domains probably modulate equilibrium between states of ACT domains.
Keywords: Abbreviations; AHAS; acetohydroxyacid synthase; ACT domain; small regulatory ligand-binding domain whose archetypes are those of aspartate kinase chorismate mutase and the TyrA protein; LSU; large subunit; SSU; small subunit; Val; r; valine-resistant phenotype; PCR; polymerase chain reaction; REM-ivrs; random extensive mutagenesis with; in vivo; reconstitution and selectionAcetohydroxyacid synthase; ACT domain; Ligand-binding domain; Subunits; Regulatory site; Random mutagenesis; Valine resistance; Reconstitution
Protein biochemistry: Don't forget the cell biology
by Gregory L. Blatch; Jude M. Przyborski (pp. 456-456).
Erratum to “Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae” [Biochim. Biophys. Acta 1804 (2010) 1516–1525]
by Francesca Guidi; Francesca Magherini; Tania Gamberi; Marina Borro; Maurizio Simmaco; Alessandra Modesti (pp. 458-458).