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BBA - Proteins and Proteomics (v.1764, #7)
Aminotryptophan-containing barstar: Structure–function tradeoff in protein design and engineering with an expanded genetic code by Marina Rubini; Sandra Lepthien; Ralph Golbik; Nediljko Budisa / (pp. 1147-1158).
The indole ring of the canonical amino acid tryptophan (Trp) possesses distinguished features, such as sterical bulk, hydrophobicity and the nitrogen atom which is capable of acting as a hydrogen bond donor. The introduction of an amino group into the indole moiety of Trp yields the structural analogs 4-aminotryptophan ((4-NH2)Trp) and 5-aminotryptophan ((5-NH2)Trp). Their hydrophobicity and spectral properties are substantially different when compared to those of Trp. They resemble the purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. The Trp → aminotryptophan substitution in proteins during ribosomal translation is expected to result in related protein variants that acquire these features. These expectations have been fulfilled by incorporating (4-NH2)Trp and (5-NH2)Trp into barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens. The crystal structure of (4-NH2)Trp-barstar is similar to that of the parent protein, whereas its spectral and thermodynamic behavior is found to be remarkably different. The Tm value of (4-NH2)Trp- and (5-NH2)Trp-barstar is lowered by about 20 °C, and they exhibit a strongly reduced unfolding cooperativity and substantial loss of free energy in folding. Furthermore, folding kinetic study of (4-NH2)Trp-barstar revealed that the denatured state is even preferred over native one. The combination of structural and thermodynamic analyses clearly shows how structures of substituted barstar display a typical structure–function tradeoff: the acquirement of unique pH-sensitive charge transfer as a novel function is achieved at the expense of protein stability. These findings provide a new insight into the evolution of the amino acid repertoire of the universal genetic code and highlight possible problems regarding protein engineering and design by using an expanded genetic code.
Keywords: Aminotryptophan; Barstar; Genetic code; Protein folding; Stability
Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01 by So-Young Jun; Shinya Fushinobu; Hideaki Nojiri; Toshio Omori; Hirofumi Shoun; Takayoshi Wakagi (pp. 1159-1166).
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the kcat/ Km values of the single mutants were 4.2–11 fold higher than that of the wild type enzyme and 1.8–4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest kcat/ Km value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 Å resolution, enabling location of the Oγ atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The kcat values of Ser34 mutants were decreased 2.9–65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state ( gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential “triad�, being intact.
Keywords: Site-directed mutagenesis; X-ray crystallography; Meta; -cleavage product hydrolase; Substrate specificity; Cumene degradation; Polychlorinated biphenyl degradation
Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus by Maw-Sheng Yeh; Ling-Rong Kao; Chang-Jen Huang; Inn-Ho Tsai (pp. 1167-1178).
To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified. TGH is an abundant homodimeric cytosolic protein with 84.2Â kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine into succinyl casein upon activation by CaCl2 in vitro. IC50 for the activation was 3Â mM, which is below the shrimp plasma Ca2+ level. Showing similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II (AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could not be isolated, their regulated expression was discussed.
Keywords: Abbreviations; Mj; Marsupenaeus japonicus; Pm; Penaeus monodon; TGH; hemocyte transglutaminase; RFE; relative fluorescence enhancementTransglutaminase; Hemolymph coagulation; Regulation by Ca; 2+; cDNA sequence; Shrimp (; Penaeus monodon; and; Marsupenaeus japonicus; )
On the statistical analysis of the GS-NS0 cell proteome: Imputation, clustering and variability testing by Norhaiza Ahmad; Jian Zhang; Phillip J. Brown; David C. James; John R. Birch; Andrew J. Racher; C. Mark Smales (pp. 1179-1187).
We have undertaken two-dimensional gel electrophoresis proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of gel-based experiments not all protein spots are detected across all samples in an experiment, and hence datasets are invariably incomplete. New approaches are therefore required for the analysis of such graduated datasets. We approached this problem in two ways. Firstly, we applied a missing value imputation technique to calculate missing data points. Secondly, we combined a singular value decomposition based hierarchical clustering with the expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have shown that while imputation of missing data was a useful method to improve the statistical analysis of such data sets, this was of limited use in differentiating between the samples investigated, and highlighted a small number of candidate proteins for further investigation.
Keywords: 2D-PAGE; proteomic profiling; NS0 cells; imputed values; hierarchical clustering; rank correlation
Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: An FT-IR study by Klaus V. Kilimann; Wolfgang Doster; Rudi F. Vogel; Christoph Hartmann; Michael G. Gänzle (pp. 1188-1197).
The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in2H2O or2H2O with 1. 5 M sucrose in the range of 6–75 °C. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The1H/2H exchange in proteins, and secondary structure elements were detected by the analysis of amide I′, amide II and amide II′ bands. A reduced1H/2H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the1H/2H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.
Keywords: FT-IR spectroscopy; Bacterial inactivation; Protein denaturation; Heat resistance
The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions by Yan-Huai R. Ding; Kim K. Hixson; Carol S. Giometti; Ann Stanley; Abraham Esteve-Núñez; Tripti Khare; Sandra L. Tollaksen; Wenhong Zhu; Joshua N. Adkins; Mary S. Lipton; Richard D. Smith; Tünde Mester; Derek R. Lovley (pp. 1198-1206).
The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.
Keywords: Proteome; Geobacter; Fe(III) reduction; Cytochrome; Anaerobic bacterium
Characterizing the specificity of activated Factor XIII for glutamine-containing substrate peptides by David B. Cleary; Muriel C. Maurer (pp. 1207-1217).
Activated Factor XIII (FXIIIa) is a transglutaminase that catalyzes the formation of γ-glutamyl-ɛ-lysine crosslinks in the fibrin network. To better understand the source of FXIIIa substrate specificity, Q-containing substrates based on β-casein, K9-peptide, and α2-antiplasmin were characterized. α2AP (1–15, Q2, Q4) and α2AP (1–15, Q2, Q4N, K12R) are highly promising peptide models since they exhibited kcat/ Km values comparable to intact β-casein. In the absence of a lysine-like donor, FXIIIa could promote deamidation of a reactive Q to an E and solution NMR served as an effective strategy for monitoring this reaction. A tendency toward deamidation allowed greater investigations of the α2-antiplasmin based peptides. FXIIIa preferentially selects the Q2 residue for carrying out crosslinking processes. The E3 and Q4 provide supporting roles in binding. When a crosslinking reaction occurs at Q2, the Q4 position is sterically blocked from reactivity. By contrast, deamidation of Q2 to E2 allows, for the first time, observation of reactivity at Q4. The K12 position provides an additional favorable site of interaction with the FXIIIa surface. The sensitivity of α2AP (1–15, Q2, Q4) to amino acid changes at Q2, Q4, and K12 suggests the importance of individual FXIIIa subsites that are controlled by chemical environment and sterics.
Keywords: Abbreviations; FXIII; Factor XIII; FXIIIa; activated FXIII; TOCSY; total correlated spectroscopy; tr-NOESY; transferred nuclear Overhauser effect spectroscopy; K; m; Michaelis–Menten kinetic constant; k; cat; catalytic constant; k; cat; /; K; m; specificity constant; K9; K9 (1–10); K9 (K7L); K9 (1–10, K7L); α; 2; AP; α; 2; -antiplasmin (1–15, Q2, Q4); α; 2; AP (Q4N); α; 2; AP (1–15, Q2, Q4N); α; 2; AP (Q4N, K12R); α; 2; AP (1–15, Q2, Q4, K12R); α; 2; AP (Q2N); α; 2; AP (1–15, Q2N, Q4); K9(Q5E); K9 (1–10, Q5E); α; 2; AP (Q2E); α; 2; AP (1–15, Q2E, Q4); α; 2; AP (Q2E, Q4N); α; 2; AP(1–15, Q2E, Q4N); α; 2; AP (Q4E); α; 2; AP (1–15, Q2, Q4E); α; 2; AP (Q2E, Q4E); α; 2; AP (1–15, Q2E, Q4E)Factor XIII; Transglutaminase; Coagulation; Deamidation; NMR; Kinetic
Cu(II) induces small-size aggregates with amyloid characteristics in two alleles of recombinant ovine prion proteins by Kirill Tsiroulnikov; Human Rezaei; Michèle Dalgalarrondo; Jean-Marc Chobert; Jeanne Grosclaude; Thomas Haertlé (pp. 1218-1226).
One of symptoms of transmissible spongiform encephalopathies is associated with the transformation of normal cellular prion protein, PrP, in its amyloid isoform resistant to proteolytic cleavage. The present study shows that interaction with copper ions converts both monomeric recombinant scrapie-susceptible PrP-VRQ and scrapie-resistant PrP-ARR variants into protease-resistant soluble oligomers with amyloid characteristics — dominant β-sheet secondary structure and interaction with thioflavine S. In contrast, binding of zinc ions resulting in the same resistance to proteolysis does not provoke transformation of α-helical monomeric structure of both PrP polymorphic variants. Cleavage of PrP N-terminus destabilises soluble form of such aggregates, and N-truncated PrPrec complexed with metal cations precipitate. N-truncated PrPrec complexed with Zn precipitated much faster than N-truncated PrPrec complexed with Cu. According to the hypothesis about the key role of small PrP oligomers in PrPC-PrPSc transformation, formation of soluble oligomers of PrP complexed with Cu can constitute an additional element in TSE propagation. Identical metal-chelating behaviour of two studied polymorphic PrPrec variants conferring different susceptibilities of sheep to scrapie could indicate their different capabilities to form fibrils. This could imply also that other factors than physico-chemical differences between PrP-VRQ and PrP-ARR and the differences in PrP transformation are responsible for the onset of TSE.
Keywords: Abbreviations; TSE; transmissible spongiform encephalopathy; PrP; prion protein; PrP; C; cellular isoform of PrP; PrP; Sc; scrapie isoform of PrP; hPrP; human PrP; PrPrec; recombinant sheep PrP; PrP-ARR and PrP-VRQ; polymorphic variants PrP-A136R154R171 and PrP-V136R154Q171; PK; proteinase K; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; CD; circular dichroism; DLS; dynamic light scattering; MOPS; 3-(; N; -morpholino) propanesulfonic acid; Tris; tris; (hydroxymethyl) aminomethane; EDTA; ethylenediaminetetraacetic acid; ThS; thioflavin SPrion protein; Amyloid; Metal ions; Copper; Zinc; Hydrolysis
The pH-dependence of amide chemical shift of Asp/Glu reflects its p Ka in intrinsically disordered proteins with only local interactions by Mario Pujato; Abel Navarro; Rodney Versace; Romina Mancusso; Ranajeet Ghose; MarÃa Luisa Tasayco (pp. 1227-1233).
Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1–37/38–108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from1H15N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293–3302]. The1H15N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH -midpoint (pHm) can be obtained by fitting. In segment A, the pHm of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the p Ka). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pHm of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pHm of its backbone amide is larger than in the p Ka of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pHm of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its p Ka whenever there are none or few neighboring backbone amides with similar pH-dependence.
Keywords: p; K; a; Nuclear magnetic resonance; Unfolded state of proteins
Compatible solutes as protectants for zymogens against proteolysis by Sonja Kolp; Markus Pietsch; Erwin A. Galinski; Michael Gütschow (pp. 1234-1242).
Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800Â mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4Â h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.
Keywords: Protein folding; Serine proteases; Zymogens; Proteolytic activation; Compatible solutes; Protein stabilization
Proton exchange coupled to the specific binding of alkylsulfonates to serum albumins by Henrik Lund; Bitten Plesner Christensen; Anders Dybdal Nielsen; Peter Westh (pp. 1243-1251).
We have applied isothermal titration calorimetry to investigate the linkage between ligand binding and the uptake or release of protons by human serum albumin (HSA) and bovine serum albumin (BSA). The ligands were sodium decyl sulfate (SDeS) and sodium dodecyl sulfate (SDS). Within a certain temperature range, the binding isotherm could be clearly resolved into two classes of sites (high affinity and low affinity) and modeled assuming independence and thermodynamic equivalence of the sites within each class. Measurements at pH 7.0 in different buffer systems revealed that the binding of SDS to the high affinity sites did not couple to any exchange of protons in either of the proteins. Saturation of the 6–8 low affinity sites for SDS, on the other hand, brought about the release of two protons from both HSA and BSA. In addition to elucidating the pH dependence of ligand binding, this analysis stressed that binding enthalpies for the low affinity sites measured by calorimetry must be corrected for effects due to the concomitant protonation of the buffer. The shorter ligand SDeS bound to HSA with a comparable stoichiometry but with four times lower affinity. Interestingly, no proton linkage was observed for the binding of SDeS. An empirical structural analysis suggested that His 242 in site 7 (of HSA) is a likely candidate for one of the proton donors.
Keywords: HSA; BSA; Calorimetry; Proton linkage; Dodecyl sulfate; Decyl sulfate
The GCN4 bZIP can bind to noncognate gene regulatory sequences by Anna V. Fedorova; I-San Chan; Jumi A. Shin (pp. 1252-1259).
We show that a minimalist basic region/leucine zipper (bZIP) hybrid, comprising the yeast GCN4 basic region and C/EBP leucine zipper, can target mammalian and other gene regulatory sequences naturally targeted by other bZIP and basic/helix–loop–helix (bHLH) proteins. We previously reported that this hybrid, wt bZIP, is capable of sequence-specific, high-affinity binding of DNA comparable to that of native GCN4 to the cognate AP-1 and CRE DNA sites. In this work, we used DNase I footprinting and electrophoretic mobility shift assay to show that wt bZIP can also specifically target noncognate gene regulatory sequences: C/EBP (CCAAT/enhancer binding protein, 5′-TTGCGCAA), XRE1 (Xenobiotic response element, 5′-TTGCGTGA), HRE (HIF response element, 5′-GCACGTAG), and the E-box (Enhancer box, 5′-CACGTG). Although wt bZIP still targets AP-1 with strongest affinity, both DNA-binding specificity and affinity are maintained with wt bZIP binding to noncognate gene regulatory sequences: the dissociation constant for wt bZIP in complex with AP-1 is 13 nM, while that for C/EBP is 120 nM, XRE1 240 nM, and E-box and HRE are in the μM range. These results demonstrate that the bZIP possesses the versatility to bind various sequences with varying affinities, illustrating the potential to fine-tune a designed protein's affinity for its DNA target. Thus, the bZIP scaffold may be a powerful tool in design of small, α-helical proteins with desired DNA recognition properties.
Keywords: bZIP; GCN4; C/EBP; Xenobiotic response element XRE1; Enhancer box E-box; HIF response element HRE
Hofmeister effects in protein unfolding kinetics: Estimation of changes in surface area upon formation of the transition state by Leticia López-Arenas; Silvia SolÃs-Mendiola; Jaqueline Padilla-Zúñiga; Andrés Hernández-Arana (pp. 1260-1267).
We studied the effect of three electrolytes (LiCl, Na2SO4, GuHCl) on the unfolding reaction of chymopapain, a two-domain protein belonging in the papain family of cysteine proteinases. Due to methodological reasons, these studies were carried out at pH 1.5 where the protein unfolds following biphasic kinetics. We have observed the presence of two different effects of electrolyte concentration on the unfolding reactions. At low ionic strength, the ionic atmosphere brought about an increase in reaction rates, regardless of the type of ions being present; this effect is attributed to a general “electrostatic screening� of charge–charge interactions in the macromolecule. At high ionic strength, each electrolyte exerted a distinctively different effect: both rate constants were largely increased by GuHCl (a well-known protein denaturant), but only slightly by LiCl; in contrast, Na2SO4 (a good precipitant) decreased the value of both unfolding rates. These ion-specific (Hofmeister) effects were further used to estimate changes in accessible surface area (ΔASA) upon formation of the transition states (TS) for unfolding. Results obtained with LiCl and Na2SO4, which we analyzed by means of a parameterization derived from published solubility data of amino acid derivatives, are consistent with ΔASA increments (for each phase) of about 8.0% of the total theoretical ΔASA for complete unfolding of the chymopapain molecule. Results in the presence of GuHCl, which were analyzed by using a previous parameterization of protein unfolding data, gave larger ΔASAs of activation, equivalent to 13 and 16% of the total unfolding ΔASA.
Keywords: Abbreviations; ANS; 8-Anilino-1-naphthalene sulfonic acid; DC; circular dichroism; TS; transition state; ΔASA; changes in accessible surface areaTransition state; Unfolding reaction; Hofmeister effect; Salt effect; Electrostatic screening
Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C–H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site by T.V. Demidkina; N.G. Faleev; A.I. Papisova; N.P. Bazhulina; V.V. Kulikova; P.D. Gollnick; R.S. Phillips (pp. 1268-1276).
In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied.The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition ofl-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C–α-proton of a number of amino acids for deuterium in2H2O. The kex values for the isotope exchange ofl-phenylalanine and 3-fluoro-l-tyrosine are close to the kcat values for reacting substrates. Thus, for the mutant TPLs the stage of C–α-proton abstraction may be considered as a rate-limiting for the whole reaction.
Keywords: Citrobacter freundii; tyrosine phenol-lyase; Asp214; Mutant enzymes; Ionic form of the internal aldimine; Steady-state kinetic; Rate-limiting stage
Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond by Gabriel Žoldák; Erik Sedlák; Eva ValuÅ¡ová; Alexandra Wolfrum; Jozef Marek; Marián AntalÃk; Mathias Sprinzl (pp. 1277-1285).
The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, ∼530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be ∼21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, Ttrs, independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, Ea, and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min−1, indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.
Keywords: Abbreviations; ANS; 1-anilinonaphthalene-8-sulphonate; EF-Ts; elongation factor Ts; EF-Ts(wt); EF-Ts wild type; EF-Ts(C190A); EF-Ts mutant with alanine substituted for cysteine 190; DSC; differential scanning calorimetry; CD; circular dichroism; T; trs; the temperature of transition of heat denaturation; GdmHCl; guanidium hydrochloride; T*; the temperature at which the rate of the irreversible step is 1 min; −1Irreversible denaturation; Residual structure; Denatured state; Intermonomeric disulfide bond; Thermus thermophilus
Conformational stability of secretory leucocyte protease inhibitor: A protein with no hydrophobic core and very little secondary structure by Curtis C.-J. Lin; Bao-Yuan Lu; Jui-Yoa Chang (pp. 1286-1291).
Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine α-chymotrypsin. The EMBO J. 7, 345–352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (Δ GH20) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.
Keywords: Abbreviations; SLPI; secretory leucocyte protease inhibitor; DTT; reduced dithiothreitol; HPLC; high pressure liquid chromatography; MALDI-TOF; matrix assisted laser desorption ionization/time-of flight; N-isomer; native protein; X-isomer; fully oxidized scrambled isomerConformational stability of SLPI; Denaturation of SLPI; Unfolding of SLPI; Method of disulfide scrambling; Scrambled isomers of SLPI; Reductive unfolding of native SLPI; Reductive unfolding of scrambled SLPI
Identification of chitotriosidase isoforms in plasma of Gaucher disease patients by two dimensional gel electrophoresis by LucÃa Quintana; Alberto Monasterio; Kepa Escuredo; Jokin del Amo; Pilar Alfonso; Felix Elortza; Simon Santa Cruz; Laureano Simón; Antonio MartÃnez; Pilar Giraldo; Miguel PocovÃ; José Luis Castrillo (pp. 1292-1298).
Chitotriosidase protein (ChT) is the most important biochemical marker described for Gaucher disease (GD). ChT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring ChT activity. Applying two-dimensional gel electrophoresis (2-DE) technology this study describes the localization and identification of five ChT isoforms in 2-DE images obtained from plasma of GD patients. All these isoforms were unequivocally identified using MALDI-TOF mass spectrometry (MS) and validated by western blot analysis. The features of each ChT isoform separated by 2-DE in plasma from GD patients homozygous for the wild-type ChT allele, carriers of one defective allele and patients homozygous for the mutant allele are presented. We also show the correlation between each ChT isoform and the plasma ChT enzymatic activity of the GD patients sampled in this study.
Keywords: Abbreviations; GD; Gaucher disease; ChT; chitotriosidaseGaucher disease; Chitotriosidase; Biomarker; 2-DE
Streptococcus gordonii soluble inorganic pyrophosphatase: An important role for the interdomain region in enzyme activity by Mohammad Ilias; Tom W. Young (pp. 1299-1306).
Streptococcus gordonii DL1(Challis) soluble inorganic pyrophosphatase was shown to be a homo dimer with a subunit molecular mass of 33407. In solution, in the presence of Mn2+, the protein is ellipsoidal with an axial ratio of 3.37 and molecular mass of 67000. In the absence of the divalent cation, the molecular mass is unchanged but the axial ratio increases to 3.94. The enzyme, in the presence of 5 mM Mg2+, at 25 °C and pH 9.0, has Km and kcat values of 62 μM and 6290 s−1, respectively. The free N- and C-terminal domains of Streptococcus gordonii PPase did not interact productively when mixed together. Replacing the interdomain region with that from Bacillus subtilis decreased the catalytic efficiency of the enzyme whereas inserting the same region from the Archaeglobus fulgidus thermophilic enzyme yielded an inactive protein. Substitution, deletion and insertion of amino acid residues in the interdomain region were found to affect the monomer dimer equilibrium in the absence of Mn2+ ions. In the presence of these ions however the variant proteins were dimers. Proteins with altered interdomain regions also displayed a 2- to 625-fold decrease in catalytic efficiency. These data together with that of computer analysis show that the interdomain region has characteristics of a mechanical hinge. Modelling mutant proteins onto the wild type shows that the active site regions are not significantly perturbed. These results show that, although distant from the active site, the interdomain region plays a role in enzyme activity and both its length and composition are important. This supports the hypothesis that catalytic activity requires the N- and C terminal domains of the enzyme to open and close using the interdomain region as a hinge.
Keywords: Inorganic pyrophosphatase Family II; Streptococcus gordonii; Chimeric and mutant proteins; Catalytic activity; Interdomain region