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BBA - Proteins and Proteomics (v.1824, #6)

Editorial Board (pp. i).

Water mobility, denaturation and the glass transition in proteins by David Porter; Fritz Vollrath (pp. 785-791).

A quantitative mechanism is presented that links protein denaturation and the protein–water glass transition through an energy criterion for the onset of mobility of strong protein–water bonds. Differences in the zero point vibrational energy in the ordered and disordered bonded states allow direct prediction of the two transition temperatures. While the onset of water mobility induces the same change in heat capacity for both transitions, the order–disorder transition of denaturation also predicts the observed excess enthalpy gain. The kinetics of the water and protein components through the glass transition are predicted and compared with dielectric spectroscopy observations. The energetic approach provides a consistent mechanism for processes such as refolding and aggregation of proteins involved in protein maintenance and adaptability, as the conformational constraints of strong water–amide bonds are lost with increased molecular mobility. Moreover, we suggest that the ordered state of peptide–water bonds is induced at the point of protein synthesis and could play a key role in the function of proteins through the enhancement of electronic activity by ferroelectric domains in the protein hydration shell, which is lost upon denaturation.► Denaturation and the protein–water glass transition are linked quantitatively. ► The instability transitions have the same elastic instability criterion in energy. ► The transitions are different in their zero point vibrational energy. ► The transitions are predicted ab initio using density functional theory. ► Thermodynamics and kinetics of the transitions are predicted.

Keywords: Protein hydration; Denaturation; Glass transition; Lysozyme; Myoglobin; DFT

Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity by Galina A. Petkova; Zaruba Kamil Záruba; Kral Vladimír Král (pp. 792-801).

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60–70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-( p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V_max and K_m) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.Display Omitted► Alcohol dehydrogenases and cofactor were immobilized on silica particles. ► Frequency shift of amide I and amide II vibrational bands of immobilized enzymes. ► Changes in enzymes secondary and tertiary structures during immobilization. ► Switch in stereoselectivity of immobilized enzyme.

Keywords: Silica nanoparticle; Alcohol dehydrogenase; Immobilization; Spectroscopy; Unfolding; Switch in enzyme stereoselectivity

Cloning, expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions by James A. Langston; Kimberly Brown; Feng Xu; Kim Borch; Ashley Garner; Matt D. Sweeney (pp. 802-812).

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene ( cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.

Keywords: Abbreviations; 2D-GE; two-dimensional gel electrophoresis; CBM; carbohydrate binding module; CDH; cellobiose dehydrogenase; CE; carbohydrate esterase; DCPIP; 2,6-dichlorophenol-indophenol; DSC; differential scanning calorimetry; FAD; flavine adenine dinucleotide; GH; glycoside hydrolase; GH61; glycoside hydrolase family 61 protein; IEF; isoelectric focusing; PASC; phosphoric acid-swollen cellulose; PDA; potato dextrose agar; PHBAH; p; -hydroxybenzoic acid hydrazideCellobiose dehydrogenase; Thielavia terrestris; Glycoside hydrolase family 61

Acclimation increases freezing stress response of Arabidopsis thaliana at proteome level by Francesca Fanucchi; Emanuele Alpi; Stefano Olivieri; Carlo V. Cannistraci; Angela Bachi; Amedeo Alpi; Massimo Alessio (pp. 813-825).

This study used 2DE to investigate how Arabidopsis thaliana modulates protein levels in response to freezing stress after sub-lethal exposure at −10°C, both in cold-acclimated and in non-acclimated plants. A map was implemented in which 62 spots, corresponding to 44 proteins, were identified. Twenty-two spots were modulated upon treatments, and the corresponding proteins proved to be related to photosynthesis, energy metabolism, and stress response. Proteins demonstrated differences between control and acclimation conditions. Most of the acclimation-responsive proteins were either not further modulated or they were down-modulated by freezing treatment, indicating that the levels reached during acclimation were sufficient to deal with freezing. Anabolic metabolism appeared to be down-regulated in favor of catabolic metabolism. Acclimated plants and plants submitted to freezing after acclimation showed greater reciprocal similarity in protein profiles than either showed when compared both to control plants and to plants frozen without acclimation. The response of non-acclimated plants was aimed at re-modulating photosynthetic apparatus activity, and at increasing the levels of proteins with antioxidant-, molecular chaperone-, or post-transcriptional regulative functions. These changes, even less effective than the acclimation strategy, might allow the injured plastids to minimize the production of non-useful metabolites and might counteract photosynthetic apparatus injuries.Display Omitted► Freezing tolerance in acclimated and non-acclimated Arabidopsis thaliana was analyzed. ► Previous acclimation gives more resistance to the subsequent freezing stress. ► Functional modulation of photosynthetic apparatus is showed. ► Down-regulation of anabolic proteins is involved in the survival strategy. ► When not acclimated, plants show weaker response.

Keywords: Arabidopsis thaliana; Freezing stress; 2D electrophoresis; Mass spectrometry

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein by Derek R. Dee; Amar Nath Gupta; Max Anikovskiy; Iveta Sosova; Elena Grandi; Laura Rivera; Abhilash Vincent; Angela M. Brigley; Nils O. Petersen; Michael T. Woodside (pp. 826-832).

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90–232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al3⁺-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al3⁺, Zn2⁺, and free-base). Buffer ionic strength also affected the binding, with K_d increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4–5 PcTS molecules per PrP molecule.► FB–, Al³⁺– and Zn²⁺–PcTS bind PrP^C(90–232) with low μM affinity. ► Al³⁺– and Zn²⁺–PcTS bind PrP^C(90–232) with a stoichiometry of 4–5 ligands/protein. ► Increasing K_d correlates with decreased anti–scrapie potency. ► PcTS binding to PrP is likely driven by interactions with aromatic residues. ► Electrostatic interactions can modulate PcTS–PrP^C binding affinity.

Keywords: Abbreviations; PcTS; phthalocyanine tetrasulfonate; FB-PcTS; free-base-phthalocyanine tetrasulfonate; PrP; ^C; natively-folded prion protein; PrP; ^Sc; misfolded, disease-related prion protein; SPR; surface plasmon resonance; ITC; isothermal titration calorimetry; FCS; fluorescence correlation spectroscopyPrion protein; Phthalocyanine tetrasulfonate; Ligand binding; ITC; SPR; Fluorescence correlation spectroscopy

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells by Jean-Paul Lasserre; Fred Fack; Tommaso Serchi; Dominique Revets; Sébastien Planchon; Jenny Renaut; Lucien Hoffmann; Arno C. Gutleb; Claude P. Muller; Torsten Bohn (pp. 833-841).

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-β estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.Display Omitted► Atrazine and PCB153 altered protein expression of MCF-7 cells during 36h exposure. ► Both cytosolic and membrane proteins were differentially regulated. ► Proteins differentially regulated were structural and hormonal regulating proteins. ► Atrazine and PCB153 act in different cellular compartments via various pathways.

Keywords: Abbreviations; PCBs; polychlorinated biphenyls; ATR; atrazine; US-EPA; US Environmental Protection Agency; E2; estradiol; ROS; reactive oxygen species; 2D-DiGE; 2D difference in gel electrophoresis; EDC; endocrine disrupting compoundsAtrazine; PCB 153; MCF-7 human cell; 2D-DiGE; Subcellular fraction

Single-shot NMR measurement of protein unfolding landscapes by Enrico Rennella; Alessandra Corazza; Luca Codutti; Araldo Causero; Vittorio Bellotti; Monica Stoppini; Paolo Viglino; Federico Fogolari; Gennaro Esposito (pp. 842-849).

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine—Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is¹⁵N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein β₂-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.Display Omitted► NMR hydrogen–deuterium exchange measurements to study protein unfolding landscape. ► BLUU-Tramp, new NMR method to measure isotope exchange thermal kinetics. ► BLUU-Tramp: time gain, single sample and experimental session, routine instruments. ► Identification of stability determinants in proteins and protein complexes. ► Application to amyloidogenic systems, physiological milieu, basic folding studies.

Keywords: Abbreviations; β2m; β; ₂; -microglobulin; BIC; Bayes Information Criterion; BLUU-Tramp; Biophysics Laboratory University of Udine—Temperature ramp; EX1; exchange with a single rate-determining step; EX2; exchange with a double rate-determining step; EXX; exchange occurring at intermediate regime between EX1 and EX2; HX; hydrogen exchange; IR; infrared; NMR; nuclear magnetic resonance; SOFAST-HMQC; band-Selective Optimized-Flip-Angle Short-Transient Heteronuclear Multiple Quantum CorrelationProtein unfolding; Energy landscape; H–D exchange; Protein NMR; Unfolding thermodynamics; Protein thermal denaturation

Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase by Elvira Romero; Michael Fedkenheuer; Samuel W. Chocklett; Jun Qi; Michelle Oppenheimer; Pablo Sobrado (pp. 850-857).

Aspergillus fumigatus siderophore A ( Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N⁵-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP⁺, and not NAD⁺, protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP⁺ to also play a role in stabilization of the C4a-hydroperoxyflavin.Display Omitted ► Af SidA utilizes both NADPH and NADH for flavin reduction. ► NADP⁺ induces conformational changes, not observed with NAD⁺. ► These conformational changes are involved in stabilization of flavin intermediates.

Keywords: SidA; Flavin-dependent monooxygenase; Aspergillus fumigatus; N; -hydroxylating; C4a-hydroperoxyflavin; Anti-fungal drug target

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BBA - Proteins and Proteomics (v.1824, #6)