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BBA - Proteins and Proteomics (v.1824, #3)

Editorial Board (pp. i).

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins by R. Shyama Prasad Rao; Moller Ian Max Møller (pp. 405-412).

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~100,281) in a large set of eukaryotic proteins (~22,995). Phosphorylation probability was found to be much higher in both the termini of protein sequences and this is much pronounced in transmembrane proteins. A large proportion (51.3%) of occupied sites had a nearby phosphorylation within a distance of 10 amino acids; however, this proportion is very high compared to the expected one (16.9%). The distribution of phosphorylated sites in proteins showed a strong deviation from the expected maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.Display Omitted► Phosphorylation occupancy is higher in both the termini of protein sequences. ► The majority of phosphorylated sites occur in close proximity of each other. ► Distribution probabilities of phosphorylated sites show a highly non-random nature. ► More than 50% of phosphorylated sites fall within a small number (40) of motifs.

Keywords: Abbreviations; GRAVY; Grand Average Hydropathy; RDF; Resource Description FrameworkMotif; Occupancy; Phosphoprotein; Phosphorylation; Probability

Crystal structure and characterization of coiled-coil domain of the transient receptor potential channel PKD2L1 by Katrina L. Molland; Lake N. Paul; Dinesh A. Yernool (pp. 413-421).

The cation-permeable channel PKD2L1 forms a homomeric assembly as well as heteromeric associations with both PKD1 and PKD1L3, with the cytoplasmic regulatory domain (CRD) of PKD2L1 often playing a role in assembly and/or function. Our previous work indicated that the isolated PKD2L1 CRD assembles as a trimer in a manner dependent on the presence of a proposed oligomerization domain. Herein we describe the 2.7Å crystal structure of a segment containing the PKD2L1 oligomerization domain which indicates that trimerization is driven by the β-branched residues at the first and fourth positions of a heptad repeat (commonly referred to as “a” and “d”) and by a conserved R–h–x–x–h–E salt bridge motif that is largely unique to parallel trimeric coiled coils. Further analysis of the PKD2L1 CRD indicates that trimeric association is sufficiently strong that no other species are present in solution in an analytical ultracentrifugation experiment at the lowest measurable concentration of 750nM. Conversely, mutation of the “a” and “d” residues leads to formation of an exclusively monomeric species, independent of concentration. Although both monomeric and WT CRDs are stable in solution and bind calcium with 0.9μM affinity, circular dichroism studies reveal that the monomer loses 25% more α-helical content than WT when stripped of this ligand, suggesting that the CRD structure is stabilized by trimerization in the ligand-free state. This stability could play a role in the function of the full-length complex, indicating that trimerization may be important for both homo- and possibly heteromeric assemblies of PKD2L1.► Crystal structure of a segment of the PKD2L1 cytoplasmic regulatory domain (CRD). ► Structure reveals a trimeric coiled coil in the predicted oligomerization region. ► Trimerization driven by central β-branched residues and stabilized by a salt bridge. ► Mutation of central residues converts high-affinity trimeric CRD to monomer. ► Monomer binds Ca²⁺ with WT-affinity, loses significant α-helicity upon ligand removal.

Keywords: Abbreviations; CRD; cytoplasmic regulatory domain; PKD2L1; polycystic kidney disease 2-like 1; PKD1; polycystic kidney disease 1; PKD1L3; polycystic kidney disease 1-like 3; ER; endoplasmic reticulum; HEK; human embryonic kidney; ADPKD; autosomal dominant polycystic kidney disease; TEV; tobacco etch virus; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MALDI-MS; matrix-assisted laser desorption/ionization mass spectroscopy; CD; circular dichroism; EDTA; ethylenediaminetetracetic acid; HBSS; HEPES-buffered saline solution; CC2; coiled-coil 2PKD2L1; Coiled-coil; Trimerization

Crystal structure of D-serine dehydratase from Escherichia coli by Darya V. Urusova; Michail N. Isupov; Svetlana Antonyuk; Galina S. Kachalova; Galina Obmolova; Alexei A. Vagin; Andrey A. Lebedev; Gleb P. Burenkov; Zbigniew Dauter; Hans D. Bartunik; Victor S. Lamzin; William R. Melik-Adamyan; Thomas D. Mueller; Klaus D. Schnackerz (pp. 422-432).

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5′-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5′-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279–Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

Keywords: Abbreviations; DSD; D-serine dehydratase; PLP; pyridoxal 5′-phosphate; LSD; rat liver L-serine dehydratase; OASS; O-acetylserine sulfhydrylase; TDH; L-threonine dehydratase; SR; serine racemase; TPS; tryptophan synthase; CBS; cysteine β-synthase; NMDA; N-methyl-D-aspartate; DTE; dithioerythritol; TAPS; N-Tris(hydroxymethyl)methyl-3-propane sulfonic acidD-Serine dehydratase; Pyridoxal 5′-phosphate; Type II fold; Open and closed conformation; α,β elimination

Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 by Stefania Montersino; Willem J.H. van Berkel (pp. 433-442).

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme.► Flavoprotein hydroxylases from R. jostii RHA1 have been functionally annotated. ► Mis-annotated Q0SFK6 encodes for 3-hydroxybenzoate 6-hydroxylase (3HB6H). ► 3HB6H is a homodimer. ► 3HB6H converts ortho- and meta-substituted 3-hydroxybenzoates. ► Gentisate is a good effector of 3HB6H.

Keywords: Fingerprint; Flavoprotein; Functional annotation; 3-hydroxybenzoate 6-hydroxylase; Monooxygenase; Rhodococcus jostii; RHA1

NMR for direct determination of K_m and V_max of enzyme reactions based on the Lambert W function-analysis of progress curves by Franziska Exnowitz; Bernd Meyer; Thomas Hackl (pp. 443-449).

¹H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K_m and V_max, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis–Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K_m and V_max were fitted to obtain the experimental progress curve and resulted in K_m=28mM and V_max=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K_m=379μM and k_cat=0.04s−¹. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.Display Omitted► Direct determination of K_m and V_max from progress curves using¹H NMR spectroscopy. ► Applying Lambert W function analysis for fitting the experimental progress curves. ► NMR can easily detect and quantify in situ all reaction products and substrates. ► NMR allows the direct analysis of the stereochemistry of the products. ► NMR is a powerful tool to analyze Michaelis–Menten kinetics of enzyme reactions.

Keywords: Abbreviations; FDP; farnesyl diphosphate; (+)-GDS; (+)-germacrene D synthase¹; H NMR; Enzyme kinetics; Progress curve analysis; Lambert W function; Invertase; Germacrene D synthase

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2 by Renée Vancraenenbroeck; Evy Lobbestael; Stephen D. Weeks; Sergei V. Strelkov; Veerle Baekelandt; Jean-Marc Taymans; Marc De Maeyer (pp. 450-460).

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC,Rasofcomplex proteins) and kinase domains and a connecting COR (C-terminusofROC) domain. In contrast, the precise functions of the protein–protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR_LRRK2) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR_LRRK2 in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR_LRRK2 thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR_LRRK2 multimerization was detected via cross-linking studies. Finally, the LRR_LRRK2 clinical mutations did not influence LRR_LRRK2 secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR_LRRK2 inter- and intramolecular interactions.► Sequence analysis reveals that LRRLRRK2 contains 14 putative LRRs. ► A model of LRRLRRK2 was constructed using an LRR template assembly approach. ► Recombinant LRRLRRK2 was purified from E. coli. ► Chemical cross-linking studies revealed that LRRLRRK2 presents as a monomer. ► Clinical mutant forms of LRRLRRK2 did not influence its macromolecular properties.

Keywords: Abbreviations; BS3; bis(sulfosuccinimidyl) suberate; CD; circular dichroism; Chaps; 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate; CL; cross-linking; DDM; n-Dodecyl-β-; d; -maltopyranoside; DLS; dynamic light scattering; DTT; dithiothreitol; EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; EDTA; ethylene-diamine-tetra-acetic acid; EGS; ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); HM; homology modeling; GA; glutaraldehyde; GuHCl; guanidine hydrochloride; IEX; ion exchange; IMAC; immobilized metal ion affinity chromatography; IPTG; isopropyl-β-; d; -thiogalacto-pyranoside; LB; Luria Bertani; M; _W; molecular weight; rASA; relative accessible surface area; R; _Stokes; Stokes radius; SDS; sodium dodecyl sulfate; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; SEC; size exclusion chromatography; S-EGS; ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); S-SDA; sulfosuccinimidyl 6-(4,4′-azipentanamido) hexanoate; UV; ultravioletParkinson's disease: leucine-rich repeat kinase 2; Leucine-rich repeat; Protein purification; Homology modeling

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus by Damasio André R.L. Damásio; Liliane F.C. Ribeiro; Lucas F. Ribeiro; Gilvan P. Furtado; Fernando Segato; Fausto B.R. Almeida; Augusto C. Crivellari; Marcos S. Buckeridge; Tatiana A.C.B. Souza; Mário T. Murakami; Richard J. Ward; Rolf A. Prade; Maria L.T.M. Polizeli (pp. 461-467).

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.► A dimeric xyloglucanase was secreted in Aspergillus nidulans strain A773 (pyrG ⁸⁹). ► The XegA far-UV CD spectrum indicated a protein rich in β-sheet structures. ► The XegA thermal stability revealed transition temperature at 50.8°C and 70°C. ► The monomeric form was more active than the dimer by hydrodynamic behavior.

Keywords: Aspergillus niveus; Aspergillus nidulans; Xyloglucanase; Secretion; Circular dichroism; Xyloglucan

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity by Ali Es-haghi; Sajad Shariatizi; Azadeh Ebrahim-Habibi; Mohsen Nemat-Gorgani (pp. 468-477).

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.► Amyloid fibrillation of chemically-modified forms of bovine carbonic anhydrase II. ► Amyloid fibrillation of bovine carbonic anhydrase II under native conditions. ► Amyloid fibrillation of apo and holo forms of bovine carbonic anhydrase II. ► Amyloid fibrillation in pre-molten globular form of bovine carbonic anhydrase II.

Keywords: Abbreviations; BCA II; bovine carbonic anhydrase II; PMG; pre-molten globule; MG; molten globule; ThT; thioflavin T; CD; circular dichroism; TEM; transmission electron microscopy; ANS; 1-anilino-naphthalene 8-sulfonate; DLS; dynamic light scattering; MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium BromideMetalloprotein; Pre-molten globule; Molten globule; Chemical modification; Surface hydrophobicity

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels by Alma L. Saucedo; Federico del Rio-Portilla; Cristiana Picco; Georgina Estrada; Gianfranco Prestipino; Lourival D. Possani; Muriel Delepierre; Gerardo Corzo (pp. 478-487).

The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1–C8, C2–C5, C3–C6, and C4–C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na_v channels.► A novel 3D structure of a scorpion neurotoxin was solved. ► A correct folding and disulfide pairing were observed in its recombinant form. ► The native and the recombinant neurotoxins maintained their electrophysiological properties.

Keywords: Abbreviations; TFA; trifluoroacetic acid; ESI; electrospray ionization; C.s. suffusus; Centruroides suffusus suffusus; HPLC; high performance liquid chromatography; rmsd; root mean standard deviation Centruroides suffusus suffusus; Disulfide bridge; Na; _v; 1.5; NMR; Scorpion toxin; Recombinant expression

Identification and classification of conopeptides using profile Hidden Markov Models by Silja Laht; Dominique Koua; Lauris Kaplinski; Frédérique Lisacek; Stocklin Reto Stöcklin; Maido Remm (pp. 488-492).

Conopeptides are small toxins produced by predatory marine snails of the genus Conus. They are studied with increasing intensity due to their potential in neurosciences and pharmacology. The number of existing conopeptides is estimated to be 1 million, but only about 1000 have been described to date. Thanks to new high-throughput sequencing technologies the number of known conopeptides is likely to increase exponentially in the near future. There is therefore a need for a fast and accurate computational method for identification and classification of the novel conopeptides in large data sets. 62 profile Hidden Markov Models (pHMMs) were built for prediction and classification of all described conopeptide superfamilies and families, based on the different parts of the corresponding protein sequences. These models showed very high specificity in detection of new peptides. 56 out of 62 models do not give a single false positive in a test with the entire UniProtKB/Swiss-Prot protein sequence database. Our study demonstrates the usefulness of mature peptide models for automatic classification with accuracy of 96% for the mature peptide models and 100% for the pro- and signal peptide models. Our conopeptide profile HMMs can be used for finding and annotation of new conopeptides from large datasets generated by transcriptome or genome sequencing. To our knowledge this is the first time this kind of computational method has been applied to predict all known conopeptide superfamilies and some conopeptide families.► We built 62 pHMMs for all described conopeptide superfamilies and families. ► For classification of the signal and propeptides the accuracy of pHMMs was 100%. ► For classification of the mature peptides the accuracy of pHMMs was 96%. ► Our set of pHMMs allows automatic classification of conopeptides from large datasets.

Keywords: Conotoxin; Conopeptide; Hidden Markov Model; Conopeptide superfamilies; Protein prediction

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L by Tatiana F.R. Costa; Flavia C.G. dos Reis; Ana Paula C.A. Lima (pp. 493-501).

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K_m, but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.► Brucipain/rhodesain undergoes substrate inhibition by allostery. ► Suceptibility to substrate inhibiton is regulated by temperature. ► Enzyme conformational changes regulate suceptibility to substrate inhibiton. ► Heparan sulfate prevents substrate inhibition through interaction with the enzyme. ► Enzyme downmodulation via allostery prevents fibrinogen degradation.

Keywords: Abbreviations; E64; (; l; -; trans; -Epoxysuccinyl-; l; -leucylamido-(4-guanidino)butane); Z-FR-MCA; benzyloxycarbonyl-phenylalanyl-arginine-7-amino-4-methylcoumarin hydrochloride; Z-VLR-MCA; benzyloxycarbonyl-valine–leucine–arginine-7-amino-4-methylcoumarin; GAG; glycosaminoglycan; DTT; dithiothreitol Trypanosoma brucei; Brucipain; Rhodesain; Heparan; Inhibition; Allosteric

Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study by Koji Yoshida; Karsten Vogtt; Zunbeltz Izaola; Margarita Russina; Toshio Yamaguchi; Marie-Claire Bellissent-Funel (pp. 502-510).

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations < u²> and a retention of a native-like structure — but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of < u²>. Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in < u²>. The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.► Structural and dynamic properties of β-lactoglobulin in aqueous alcohol solutions have been revealed. ► Protein aggregation state and internal motions depend on alcohol concentration. ► Internal motions are more sensitive to aggregation than the structure. ► Structure/dynamics studies are necessary to obtain a complete picture of a molecular process.

Keywords: β-Lactoglobulin; Alcohol induced change; Small-angle neutron scattering; Quasi-elastic neutron scattering

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli by Eiji Ohmae; Chiho Murakami; Shin-ichi Tate; Kunihiko Gekko; Kazumi Hata; Kazuyuki Akasaka; Chiaki Kato (pp. 511-519).

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were −45 and −53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of −77 and −85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.► We studied the effects of pressure on deep-sea DHFR of Moritella profunda (mpDHFR). ► Enzyme activity of mpDHFR was optimal at 50MPa. ► mpDHFR is resistant to heat but sensitive to pressure and urea. ► Volume change due to unfolding of mpDHFR is smaller than that of DHFR of E. coli. ► The cavity and surface hydration dominantly contribute to the stability of mpDHFR.

Keywords: Abbreviations; CD; circular dichroism; DHF; dihydrofolate; DHFR; dihydrofolate reductase; ecDHFR; Escherichia coli; DHFR; mpDHFR; Moritella profunda; DHFR; THF; tetrahydrofolateCavity; Hydration; Deep sea; Dihydrofolate reductase; Molecular adaptation; Moritella profunda

Antigen–antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures by Thiruvarangan Ramaraj; Thomas Angel; Edward A. Dratz; Algirdas J. Jesaitis; Brendan Mumey (pp. 520-532).

The structures of protein antigen–antibody (Ag–Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab–Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag–Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag–Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag–Ab crystal structures that help explain the biological range and specificity of Ag–Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.► Ag-Ab interfaces are a rich source of information about Ag folding. ► 53 non-redundant 3D structures of Ag-Ab complexes are computationally analyzed. ► Interface features, composition, interaction frequencies averaged and summarized. ► Confirms importance of paratope aromatics. ► Provides scaling of the importance of all amino acids and their interactions.

Keywords: Epitope; Paratope; Protein–antigen/antibody interface; Computational structural summary of epitopes and paratope; Specificity of interaction of epitope and paratope amino acid residue

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BBA - Proteins and Proteomics (v.1824, #3)