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BBA - Proteins and Proteomics (v.1814, #5)

Editorial Board (pp. i).

Effect of alternative distal residues on the reactivity of cytochrome c peroxidase: Properties of CcP mutants H52D, H52E, H52N, and H52Q by Miriam C. Foshay; Lidia B. Vitello; James E. Erman (pp. 525-535).

To test the effect of alternative bases at the distal histidine position, four CcP variants have been constructed that substitute the two basic residues, aspartate and glutamate, and their amides, asparagine and glutamine, for histidine-52, i.e., CcP(H52D), CcP(H52E), CcP(H52N), and CcP(H52Q). All four mutants catalyze oxidation of ferrocytochrome c by H₂O₂ with steady-state activities that are between 250 and 7700 times slower than wild-type CcP at pH 6.0, 0.10M ionic strength, 25°C. The rate of Compound I formation is decreased between 3.5 and 5.4 orders of magnitude for the mutants compared to wild-type CcP, with the rate of the reaction between CcP(H52Q) and H₂O₂ the slowest yet observed for any CcP mutant. A correlation between the rate of Compound I formation and the rate of HCN binding for CcP and various CcP distal pocket mutants provides strong evidence that the rate-limiting step in CcP Compound I formation is deprotonation of H₂O₂ within the distal heme pocket under the experimental conditions employed in this study. While CcP(H52E) reacts stoichiometrically with H₂O₂ to form Compound I, only ~36% of CcP(H52D), ~21% of CcP(H52Q) and ~8% of CcP(H52N) appear to be converted to Compound I during their respective reactions with H₂O₂. This is partially due to the slow rate of Compound I formation and the rapid endogenous decay of Compound I for these mutants. The pathways for the endogenous decay of Compound I for the four mutants used in this study are distinct from that of wild-type CcP Compound I.Display Omitted► Four CcP mutants (H52D, H52E, H52Q, H52N) have been characterized. ► The mutants retain between 0.01 and 0.4% of the catalytic activity of CcP. ► The CcP(H52Q)-H₂O₂ reaction is the slowest yet observed for any CcP mutant. ► The endogenous decay of Compound I for the mutants is distinct from that of wtCcP. ► The rates of Compound I formation and HCN binding are correlated in 9 CcP mutants.

Keywords: Cytochrome; c; peroxidase; Distal histidine; H; ₂; O; ₂; reactivity; Compound I decay; Cyanide binding

Structural characterization and unfolding mechanism of human 4F2hc ectodomain by Javier Turnay; Joana Fort; Nieves Olmo; Santiago-Gomez Angélica Santiago-Gómez; Palacin Manuel Palacín; M Antonia Lizarbe (pp. 536-544).

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57–59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M⁻¹). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)₈ barrel; C, antiparallel β₈ sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.► 4F2hc-ED is monomeric, highly compact and stable, with buried Trp residues ► Urea unfolding equilibrium reveals two intermediate states ► I1 state resembles a highly structured molten globule ►I2 state is mainly unstructured but with strong hydrophobic interactions ►Folding is probably initiated by formation of hydrophobic clusters

Keywords: CD98hc; Circular dichroism; Fluorescence spectroscopy; Molten globule; TIM barrel; Urea denaturation.

Galactonolactone oxidoreductase from Trypanosoma cruzi employs a FAD cofactor for the synthesis of vitamin C by Elena V. Kudryashova; Nicole G.H. Leferink; Ilse G.M. Slot; Willem J.H. van Berkel (pp. 545-552).

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are interesting targets for drug therapy. The terminal step in ascorbate biosynthesis is catalyzed by flavin-dependent aldonolactone oxidoreductases belonging to the vanillyl-alcohol oxidase (VAO) protein family. Here we studied the properties of recombinant T. cruzi galactonolactone oxidoreductase (TcGAL), refolded from inclusion bodies using a reverse micelles system. The refolded enzyme shows native-like secondary structure and is active with bothl-galactono-1,4-lactone andd-arabinono-1,4-lactone. At odd with an earlier claim, TcGAL employs a non-covalently bound FAD as redox-active cofactor. Moreover, it is shown for the first time that TcGAL can use molecular oxygen as electron acceptor. This is in line with the absence of a recently identified gatekeeper residue that prevents aldonolactone oxidoreductases from plants to act as oxidases.Display Omitted► Trypanosoma cruzi galactonolactone oxidoreductase was refolded in reverse micelles. ► Refolded TcGAL is active with L-galactono-1,4-lactone and D-arabinono-1,4-lactone. ► TcGAL employs FAD as cofactor for the synthesis of vitamin C. ► TcGAL can use molecular oxygen as electron acceptor.

Keywords: Abbreviations; AOT; bis(2-ethylhexyl)sulfosuccinate; AtGALDH; Arabidopsis thaliana; l; -galactono-1,4-lactone dehydrogenase; CD; circular dichroism; DCPIP; 2,6-dichlorophenolindophenol; DTT; dithiothreitol. GSH, reduced glutathione; GSSG; oxidized glutathione; RM; Reverse micelles; TcGAL; Trypanosoma cruzi; galactonolactone oxidoreductase; VAO; vanillyl-alcohol oxidaseFlavoprotein; Galactonolactone oxidoreductase; Refolding; Reverse micelles; Trypanosoma cruzi; Vitamin C

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation by S. Fabio Falsone; Gerd Leitinger; Anita Karner; Andreas J. Kungl; Simone Kosol; Roberto Cappai; Klaus Zangger (pp. 553-561).

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.► The neurotransmitter serotonin (5-HT) suppresses amyloid fibril growth of alpha-synuclein (AS). ► 5-HT binds to intermediate aggregates of alpha-synuclein, not to monomeric AS. Consequently, 5-HT does not influence initial steps of amyloidogenesis. ► 5-HT promotes the accumulation of partially structured, SDS-resistant “on pathway” aggregates of AS. ► The C-terminal region of AS is essential for a charge dependent interaction. ► “On pathway” and “off-pathway” aggregations of AS might mechanistically overlap.

Keywords: Abbreviations; AS; α-synuclein; 5-HT; serotonin; 5,7-HT; 5,7 dihydroxytryptamine; 5-HIAA; 5-hydroxyindoleacetic acid; ThioT; thioflavin T; TEM; transmission electron spectroscopy; DLS; dynamic light scattering; NAC-region; non-Aβ component regionProtein misfolding; Amyloid; Aggregation; Parkinson disease; Neurodegeneration; Indoleamine

Protein–surfactant interactions: A tale of many states by Daniel Otzen (pp. 562-591).

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denature proteins, the field has kept a relatively low profile until the last decade. Within this period there has been a maturation of techniques for more accurate and sophisticated analyses of protein–surfactant complexes such as calorimetry and small angle scattering techniques. In this review I provide an overview of different useful approaches to study these complexes and identify eight different issues which define central concepts in the field. (1) Are proteins denatured by monomeric surfactant molecules, micelles or both? (2) How does unfolding of proteins in surfactant compare with “proper” unfolding in chemical denaturants? Recent work has highlighted the role of shared micelles, rather than monomers, below the critical micelle concentration (cmc) in promoting both protein denaturation and formation of higher order structures. Kinetic studies have extended the experimentally accessible range of surfactant concentrations to far above the cmc, revealing numerous different modes of denaturation by ionic surfactants below and above the cmc which reflect micellar properties as much as protein unfolding pathways. Uncharged surfactants follow a completely different denaturation strategy involving synergy between monomers and micelles. The high affinity of charged surfactants for proteins means that unfolding pathways are generally different in surfactants versus chemical denaturants, although there are common traits. Other issues are as follows: (3) Are there non-denaturing roles for SDS? (4) How reversible is unfolding in SDS? (5) How do solvent conditions affect the way in which surfactants denature proteins? The last three issues compare SDS with “proper” membranes. (6) Do anionic surfactants such as SDS mimic biological membranes? (7) How do mixed micelles interact with globular proteins? (8) How can mixed micelles be used to measure the stability of membrane proteins? The growing efforts to understand the unique features of membrane proteins have encouraged the development of mixed micelles to study the equilibria and kinetics of this class of proteins, and traits which unite globular and membrane proteins have also emerged. These issues emphasise the amazing power of surfactants to both extend the protein conformational landscape and at the same time provide convenient and reversible short-cuts between the native and denatured state for otherwise obdurate membrane proteins.► This review summarizes advances in protein surfactant interactions since the 1990s. ► Shared micelles are key to protein denaturation below the cmc. ► Numerous micellar denaturation mechanisms occur above the cmc. ► Mixed micelles are highly useful tools to reversibly unfold membrane proteins. ► Chain length and micelle composition both modulate protein denaturation mechanisms.

Keywords: Shared micelle; Mixed micelle; Unfolding kinetics; Binding stoichiometry; Small angle X-ray scattering; Membrane protein stability

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding by Emilia Gospodarska; Anna Kupniewska-Kozak; Grazyna Goch; Michal Dadlez (pp. 592-609).

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein–ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.Display Omitted► Binding of truncated variants of Aβ peptide with V-domain of RAGE was studied. ►Minimized fragment KLVFFAED retains specific binding. ►KLVFFAED blocks the internalization of Aβ into epithelial cell culture. ►KLVFFAED provides a lead for design of an inhibitor of Aβ transport across the BBB.

Keywords: Abbreviations; AD; Alzheimer's disease; Aβ; amyloid beta; RAGE; receptor for advanced glycation endproducts; sRAGE; soluble receptor for advanced glycation endproducts; BBB; blood–brain barrier; BCA; bicinchoninic acid; CNS; central nervous system; CSF; cerebrospinal fluid; NF-κB; nuclear factor-kappa B; NMR; nuclear magnetic resonance; MS; mass spectrometry; ESI MS; electrospray ionization mass spectrometry; Q-TOF; quadrupole time-of-flight; TEV; tobacco etch virus; FPLC; fast protein liquid chromatography; RP-HPLC; reversed-phase high-performance liquid chromatographyAlzheimer's disease; Amyloid β-peptide; Receptor for advanced glycation endproducts; Mass spectrometry; Fluorescence

N-linked glycosylation of G. mellonella juvenile hormone binding protein — Comparison of recombinant mutants expressed in P. pastoris cells with native protein by Beata Winiarska; Angela Dwornik; Debski Janusz Dębski; Krystyna Grzelak; Dominika Bystranowska; Marta Zalewska; Michał Dadlez; Ozyhar Andrzej Ożyhar; Marian Kochman (pp. 610-621).

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn⁹⁴) of the two potential N-linked glycosylation sites (Asn⁴ and Asn⁹⁴). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn⁹⁴ reduces the level of protein secretion whereas mutation at the Asn⁴ site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.►JHBP molecule from G. mellonella contains two potential N-glycosylation sites. ►In G. mellonella JHBP is glycosylated only at one site among the two available in P. pastoris. ►N-linked glycosylation affects secretion and thermal stability of JHBP molecule.

Keywords: Abbreviations; JHBP; juvenile hormone binding protein; JH; juvenile hormone; ESI-MS; electrospray-ionization mass spectrometryJHBP; Glycosylation; Pichia pastoris

Characterization of the aldol condensation activity of the trans- o-hydroxybenzylidenepyruvate hydratase-aldolase ( tHBP-HA) cloned from Pseudomonas fluorescens N3 by Silvia Ferrara; Erika Mapelli; Guido Sello; Patrizia Di Gennaro (pp. 622-629).

The gene encoding trans- o-hydroxybenzylidenepyruvate hydratase-aldolase ( tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase of class I that reversibly catalyzes the transformation of the trans- o-hydroxybenzylidenepyruvate ( t-HBP), releasing pyruvate and salicylaldehyde. The enzyme was expressed in Escherichia coli as a recombinant protein of 38kDa with a His6-Tag at its N-terminus. The recombinant protein His- tHBP-HA was purified by affinity chromatography and we present here the biochemical characterization of its activity in the aldol condensation reaction. The aldol condensation reaction parameters were determined using as acceptors both salicylaldehyde, which is the natural substrate taking part to the naphthalene degradative pathway, and benzaldehyde. In both cases, His- tHBP-HA shows similar apparent K_m and apparent V_max values. Further analyses showed that the optimal pH and temperature of His- tHBP-HA activity are 7.0 and 30°C, respectively. The tHBP-HA catalytic rates and the availability of an efficient system to produce large amounts of purified protein are relevant from a biotechnological point of view.► Aldol condensation activity of the trans- o-hydroxybenzylidenepyruvate hydratase-aldolase ( tHBP-HA) cloned from Pseudomonas fluorescens N3. ► Aldol condensation reactions. ► Aldolases of class I. ► Recombinant proteins.

Keywords: Recombinant production; t; HBP-HA; Aldol condensation; Pseudomonas

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: A molecular dynamics and normal mode analysis study by Satyavani Vemparala; Sonali Mehrotra; Hemalatha Balaram (pp. 630-637).

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.► Reduced loop movements in thermophilic adenylosuccinate synthetase (AdSS). ► Switch Loop and catalytic residues pre-arranged. ► Enhanced salt bridges and stability at high temperatures in theromophilic AdSS. ► Coordinated VAL and ASP loop movements sufficient for ligand entry.

Keywords: Enzyme catalysis; MD simulations; Normal mode analysis; Loop dynamics

Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor by Karin Ernberg; Bo Zhong; Kristin Ko; Larry Miller; Yen Hoang le Nguyen; Lawrence M. Sayre; J. Mitchell Guss; Irene Lee (pp. 638-646).

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold K_m). Comparing the IC₅₀ values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site.Display Omitted► 5-phenoxy-2,3-pentadienylamine inactivates Arthrobacter globiformis amine oxidase (AGAO). ► 6-phenyl-2,3-hexadienylamine inactivates AGAO. ► Crystal structures of AGAO attached to inactivators reveal mechanism of action. ► IC₅₀ values reveal inhibition potency and suggest selectivity in AGAO inactivation. ►Structural framework to guide the design of selective mechanism-based inhibitors.

Keywords: Abbreviations; CAO; copper-containing amine oxidase; AGAO; Arthrobacter globiformis; amine oxidase; PHDA; racemic 6-; p; henyl-2,3-; h; exa; d; ienyl; a; mine; POPDA; racemic 5-; p; hen; o; xy-2,3-; p; enta; d; ienyl; a; mine; PSB; product Schiff base; SSB; substrate Schiff base; TPQ; trihydroxyphenylalanine quinoneAllenic amine; Arthrobacter globiformis; amine oxidase; Mechanism-based inactivator; Crystal structure; Inhibition potency

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens by Shih-Ping Su; Jason D. McArthur; Roger J.W. Truscott; J. Andrew Aquilina (pp. 647-656).

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0–86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.► Crystallins form a broad mixture of cross-linked species of ~40kDa in the water-soluble fractions of human lenses of all ages. ► Low molecular weight crystallin fragments were increasingly found in the water-insoluble lens fractions with age. ► Proteolysis of beaded filament proteins occurs very early in life. ► α-Crystallin interacts with intermediate filament proteins and their breakdown products under mild heat stress.

Keywords: Abbreviations; BFs; beaded filaments; CID; collision-induced dissociation; CUI; cortex urea-insoluble; CUS; cortex urea-soluble; CWS; cortex water-soluble; ESI; electrospray ionization; HRP; horseradish peroxidise; IEF; isoelectric focusing; IFs; intermediate filaments; IgG; immunoglobulin G; IPG; immobilized pH gradient; MALDI; matrix-assisted laser desorption/ionization; MBP; maltose-binding protein; MIP; major intrinsic protein; MS; mass spectrometry; NWS; nucleus water-soluble; NUI; nucleus urea-insoluble; NUS; nucleus urea-soluble; PCR; polymerase chain reaction; PMF; peptide mass fingerprinting; PTMs; posttranslational modifications; PVDF; polyvinylidene fluoride; US; urea-soluble; UI; urea-insoluble; WI; water-insoluble; WS; water-solubleLens; Crystallins; Aging; Mass spectrometry; Intermediate filaments; Beaded filaments

Identification of a novel and potent inhibitor of phospholipase A₂ in a medicinal plant: Crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A₂ complexed with berberine by D. Naveen Chandra; G.K. Prasanth; Nagendra Singh; Sanjit Kumar; O. Jithesh; C. Sadasivan; Sujatha Sharma; Tej P. Singh; M. Haridas (pp. 657-663).

Crystal of Russell Viper venom phospholipase A₂ complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A₂. Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A₂. It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA₂.► Berberine, an alkaloid found in some medicinal plants inhibits phospholipase A₂ enzyme. ► It binds to the active site of the enzyme and inhibits it competitively. ► The binding affinity is of the order of 10−⁸M between berberine and phospholipase A₂.

Keywords: Phospholipase A; ₂; inhibition; Berberine; X-ray crystallography; Surface Plasmon Resonance

TMBHMM: A frequency profile based HMM for predicting the topology of transmembrane beta barrel proteins and the exposure status of transmembrane residues by Nitesh Kumar Singh; Aaron Goodman; Peter Walter; Volkhard Helms; Sikander Hayat (pp. 664-670).

Transmembrane beta barrel (TMB) proteins are found in the outer membranes of bacteria, mitochondria and chloroplasts. TMBs are involved in a variety of functions such as mediating flux of metabolites and active transport of siderophores, enzymes and structural proteins, and in the translocation across or insertion into membranes. We present here TMBHMM, a computational method based on a hidden Markov model for predicting the structural topology of putative TMBs from sequence. In addition to predicting transmembrane strands, TMBHMM also predicts the exposure status (i.e., exposed to the membrane or hidden in the protein structure) of the residues in the transmembrane region, which is a novel feature of the TMBHMM method. Furthermore, TMBHMM can also predict the membrane residues that are not part of beta barrel forming strands. The training of the TMBHMM was performed on a non-redundant data set of 19 TMBs. The self-consistency test yielded Q₂ accuracy of 0.87, Q₃ accuracy of 0.83, Matthews correlation coefficient of 0.74 and SOV for beta strand of 0.95. In this self-consistency test the method predicted 83% of transmembrane residues with correct exposure status. On an unseen, non-redundant test data set of 10 proteins, the 2-state and 3-state TMBHMM prediction accuracies are around 73% and 72%, respectively, and are comparable to other methods from the literature. The TMBHMM web server takes an amino acid sequence or a multiple sequence alignment as an input and predicts the exposure status and the structural topology as output. The TMBHMM web server is available under the tmbhmm tab at:http://service.bioinformatik.uni-saarland.de/tmx-site/.► We present a hidden Markov model based method for the prediction of transmembrane beta barrel proteins. ► The TMBHMM also predicts the exposure status of residues predicted to be in the membrane. ► The TMBHMM method has been compared with 2 other methods from the literature. ► The TMBHMM method has also been implemented as a web server.

Keywords: Transmembrane beta barrel proteins; Hidden Markov model; Membrane protein topology; Structure prediction web server; Lipid exposure

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner by Oktay K. Gasymov; Adil R. Abduragimov; Ben J. Glasgow (pp. 671-683).

The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor–ligand interactions. A single disulfide bond, (Cys61―Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (ΔG⁰) decreases from 4.9±0.2 to 2.1±0.3kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8±0.2 to 3.3s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2±0.2 to 22.2±0.4s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitic acid (PA–TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3Å, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2Å). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and an energetically favorable ligand-protein complex.Display Omitted► Disulfide reduction promotes the plasticity for the hairpin CD. ► The time constant of binding kinetics for NBD cholesterol is decreased 3 fold. ► However, the time constant of binding kinetics for NBD C₁₂-HPC is only slightly decreased. ► Disulfide reduction promotes conformational changes to favor binding the bulky ligand.

Keywords: Abbreviations; CD; circular dichroism; DAUDA; 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid; FRET; fluorescence resonance energy transfer; 1,5-IAEDANS; 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; NATA; N-acetyl-L-tryptophanamide; NBD cholesterol; 22-(; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3u03B2-ol; NBD C; ₁₂; -HPC; 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; SDTF; site directed tryptophan fluorescence; TCEP; tris(2-Carboxyethyl) phosphine hydrochloride; TL; human tear lipocalinTear lipocalin; Ligand binding; Disulfide motive; FRET; Time-resolved fluorescence; Protein dynamic; Lipocalin-1

Construction of a fully active Cys-less elongation factor Tu: Functional role of conserved cysteine 81 by Evelina Ines De Laurentiis; Fan Mo; Hans-Joachim Wieden (pp. 684-692).

In order to study the structural and functional requirements of the essential translational GTPase elongation factor (EF) Tu for efficient and accurate ribosome-dependent protein synthesis, construction of a cysteine-free (Cys-less) mutant variant allowing for the site-directed introduction of fluorescent and non-fluorescent labels is of great importance. However, previous reports suggest that a cysteine residue in position 81 of EF-Tu from Escherichia coli is essential for its function. To study the functional role of cysteine 81 and to construct a fully active Cys-less EF-Tu, we have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitutions as well as the isosteric amino acid serine. By characterizing nucleotide binding, EF-Ts interaction, aminoacyl-tRNA binding, and delivery to the ribosome, we demonstrate that only alanine (or cysteine) can be tolerated in this position and that the serine and methionine substitutions significantly impair aminoacyl-tRNA, but not nucleotide binding. Our findings suggest a critical functional role of the amino acid residue in position 81 of EF-Tu with respect to aminoacyl-tRNA binding. Based on structural considerations, we suggest that position 81 indirectly contributes to aminoacyl-tRNA binding through the accurate positioning of helix B.► Construction of fully functional Cysteine-free (Cys-less) Elongation Factor Tu. ► Rapid-kinetics determination of rate constants governing nucleotide interaction. ► Effects on aminoacyl-tRNA binding properties. ► Highly conserved Cysteine 81 is not strictly required for EF-Ts and ribosome interaction. ► Cysteine 81 is involved in aminoacyl-tRNA binding through positioning of helix B.

Keywords: Abbreviations; aa-tRNA; aminoacyl-tRNA; EF-Tu; elongation factor Tu; FRET; fluorescence resonance energy transfer; sm; single molecule; LB; Luria–Bertani; TPCK; N; -tosyl-; l; -phenylalanylchloromethane; P; _i; inorganic phosphate; IPTG; isopropyl-β-; d; -thiogalactopyranoside; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PEP; phosphoenolpyruvate; TCA; Trichloroacetic acid; mant-GTP; 2′(3′)-O-(; N; -methyl-anthraniloyl)-guanosine-5-triphosphate, mant-GDP, 2′(3′)-O-(; N; -methyl-anthraniloyl)-guanosine-5-diphosphateProtein design; Evolution; Fluorescence; Cysteine free; Rapid kinetics; Elongation factor Tu

Intrinsically disordered proteins may escape unwanted interactions via functional misfolding by Vladimir N. Uversky (pp. 693-712).

Intrinsically disordered proteins are highly abundant in nature and play a number of crucial roles in the living cells. They are commonly involved in a wide range of intermolecular interactions, and some of them possess remarkable binding promiscuity, being able to interact specifically with structurally unrelated partners. Although they do not have well-folded structure, some IDPs are known to fold at binding to their specific partners. IDPs are highly pliable and one IDP can form an array of unrelated structures being bound to different partners. It is believed that many IDPs, being mostly disordered, have transient elements of the preformed secondary structure which are highly interaction prone and is used by IDPs for binding to specific partners. The overall disordered nature of IDPs, their high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and their ability to morph into differently-shaped bound configurations raised a very important question about the mechanisms preventing IDPs from unwanted interactions with non-native partners. In this review, a concept of functional misfolding is introduced. Accumulated to date data on the conformational behavior and fine structure of several IDPs suggest that the preformed binding elements might be involved in a set of non-native intramolecular interactions. In other words, there is a chance that a polypeptide chain misfolds to sequester the preformed elements inside the non-interactive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners.► IDPs are abundant in nature and have crucial functions. ► They possess remarkable binding promiscuity and can fold at binding to partners. ► They often have transient, interaction prone elements that are used for binding. ► Unbound IDPs can functionally misfold, thus sequestering the preformed elements. ► Functional misfolding prevents IDPs from the unwanted interactions.

Keywords: Intrinsically disordered protein; Misfolding; Partially folded protein; Protein–protein interaction; Protein non-folding; Protein function

Effect of protein–surfactant interactions on aggregation of β-lactoglobulin by Jon G. Hansted; Peter L. Wejse; Hans Bertelsen; Daniel E. Otzen (pp. 713-723).

The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alkyl maltopyranosides (xM) reduce aggregation. Sodium dodecyl sulfate (SDS) binds to non-aggregated βLG in several steps, but reduction of aggregation was associated with the first binding step, which occurs far below the critical micelle concentration. In contrast, micellar concentrations of xMs are required to reduce aggregation. The ranking order for reduction of aggregation (normalized to their tendency to self-associate) was C10–C12>C8>C14 for SxS and C8>C10>C12>C14>C16 for xM. xTAC promote aggregation in the same ranking order as xM reduce it. We conclude that SxS reduce aggregation by stabilizing the protein's ligand-bound state (the melting temperature t_m increases by up to 10°C) and altering its charge potential. xM monomers also stabilize the protein's ligand-bound state (increasing t_m up to 6°C) but in the absence of charged head groups this is not sufficient by itself to prevent aggregation. Although micelles of both anionic and non-ionic surfactants destabilize βLG, they also solubilize unfolded protein monomers, leaving them unavailable for protein–protein association and thus inhibiting aggregation. Cationic surfactants promote aggregation by a combination of destabilization and charge neutralization. The food compatible surfactant sodium dodecanoate also inhibited aggregation well below the cmc, suggesting that surfactants may be a practical way to modulate whey protein properties.Display Omitted► Aggregation of the whey protein βLG is inhibited by sub-micellar anionic surfactants. ► This involves a combination of protein stabilization and increase of charge. ► Cationic surfactants promote aggregation below the cmc but inhibit it above the cmc. ► Non-ionic surfactants inhibit aggregation above the cmc despite destabilizing βLG. ► These different interactions can modulate the properties of whey aggregates.

Keywords: Abbreviations; βLG; β-lactoglobulin; DDM; dodecyl maltoside; DM; decyl maltopyranoside; LTAC; lauryl trimethyl ammonium chloride; R; _H; hydrodynamic radius; SDS; sodium dodecyl sulfate; SxS; sodium alkyl sulfates; xM; alkyl maltopyranosides; xTAC; alkyl trimethyl ammonium chlorides; MES; 2-(; N; -morpholino)ethanesulfonic acidIsothermal titration calorimetry; Surfactant; Emulsifier; Whey protein aggregate; Sodium dodecyl sulfate; Thermal stabilization

Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments by Yoshitaka Umetsu; Takeshi Tenno; Natsuko Goda; Masahiro Shirakawa; Takahisa Ikegami; Hidekazu Hiroaki (pp. 724-730).

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe⁶, Tyr¹⁰, Leu¹³, and Met¹⁷ found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water–micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.► Structural determination of VIP-G in MeOH and in DPC micelle. ► Micelle-bound VIP-G adopts a curve α-helix. ► Four residues located at a concave face form a hydrophobic patch. ► These residues play an important role for the membrane binding.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; TRX; thioredoxin; PACAP; pituitary adenylate cyclase-activating polypeptide; VIP; vasoactive intestinal peptide; HSQC; heteronuclear single quantum coherence; DPC; dodecylphosphocholine; GPCR; G protein-coupled receptor; RA; rheumatoid arthritis; SAR; structure–activity relationship; TFE; trifluoroethanolVasoactive intestinal peptide; Pituitary adenylate cyclase-activating peptide; G protein-coupled receptor; NMR structure; Dodecylphosphocholine micelle

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BBA - Proteins and Proteomics (v.1814, #5)