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BBA - Proteins and Proteomics (v.1774, #2)

Editorial Board (pp. ii).

Annotating the human proteome: Beyond establishing a parts list by Michael Mueller; Lennart Martens; Rolf Apweiler (pp. 175-191).

The completion of the human genome has shifted the attention from deciphering the sequence to the identification and characterisation of the functional components, including genes. Improved gene prediction algorithms, together with the existing transcript and protein information, have enabled the identification of most exons in a genome. Availability of the ‘parts list’ has fostered the development of experimental approaches to systematically interrogate gene function on the genome, transcriptome and proteome level. Studying gene function at the protein level is vital to the understanding of how cells perform their functions as variations in protein isoforms and protein quantity which may underlie a change in phenotype can often not be deduced from sequence or transcript level genomics experiments alone. Recent advancements in proteomics have afforded technologies capable of measuring protein expression, post-translational modifications of these proteins, their subcellular localisation and assembly into complexes and pathways. Although an enormous amount of data already exists on the function of many human proteins, much of it is scattered over multiple resources. Public domain databases are therefore required to manage and collate this information and present it to the user community in both a human and machine readable manner. Of special importance here is the integration of heterogeneous data to facilitate the creation of resources that go beyond a mere parts list.

Keywords: Human; Proteomics; Functional annotation; Data integration; Data standardisation

Fractal aggregation of porcine fumarase induced by free radicals by Mario Barteri; Carlo Coluzza; Simona Rotella (pp. 192-199).

The present study demonstrates that H₂O₂^{and OH} cause fibril aggregation and catalytic inactivation of porcine fumarase. In the aggregated (oxidized) enzyme, modifications in both secondary and tertiary protein structure occur and the enzyme aggregation obeys to fractal geometry. We then collected information on the fractal dimension and on the size and shape of fumarase aggregates by using Synchrotron Radiation (SR) Small Angle X-ray Scattering (SAXS) analysis. The geometrical self-similarity assessment of aggregates has been revealed by both AFM and SEM measurements at different scale of magnification. Micrographs collected remarkably demonstrate that the oxidized enzyme shows dendritic fractal structure over a large range of sizes.

Keywords: Fractal aggregation; Porcine fumarase; SR-SAXS; NSAFM; SEM

Epitope characterization of ovalbumin in BALB/c mice using different entry routes by Yoshinori Mine; Marie Yang (pp. 200-212).

Ovalbumin (OVA) is known as a major allergen in egg white. A number of studies have reported the partial T and B cell epitope mapping of OVA using murine models and allergic patients' sera. Recently, we have reported the IgE-binding regions of the entire OVA molecule using egg allergic patients' sera. However, the entire epitope mapping of OVA in a murine model has not been completed yet. In the present study, BALB/c mice were administered a solution of OVA using three different entry routes (oral, intraperitoneal and subcutaneous) with their respective adjuvant (cholera toxin, aluminum hydroxide and Freund's adjuvant). Two nitrocellulose membranes containing 188 overlapping synthetic peptides (with a length of 12 amino acids and an offset of two amino acids) covering the primary sequence of OVA, were probed with the three different BALB/c mice antisera. Antisera obtained from orally challenged mice identified eight IgE epitope regions, i.e. I53D60; V77R84; S103E108; G127T136; E275V280; G301F306; I323A332 and A375S384, while sera raised by intraperitoneal and subcutaneous injections exhibited two (K55D60 and K277L282) and five (K55R58; G127T136; K279L282; T303S308 and I323A332) IgE binding sequences, respectively. The residues critical for the epitope–paratope interactions were finely characterized using the oral immunization serum. Analysis of IgE binding epitopes in mice provides us with potential strategies for design of specific immunotherapy.

Keywords: Abbreviations; APC; Antigen presenting cell; CFA; Complete Freund's adjuvant; DC; Dendritic cells; Fmoc; fluorenylmethoxycarbonyl; Ig; Immunoglobulins; IFA; Incomplete Freund's adjuvant; OVA; Ovalbumin; SPOTS; Simple Precise Original Test System; TBS; Tris-buffered saline; TBST; Tris buffered saline containing Tween-20BALB/c mouse; Immunoglobulin E epitope mapping; Ovalbumin; Route of immunization; SPOTS membrane

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase by Roshan V. Kukreja; Sapna Sharma; Shuowei Cai; Bal Ram Singh (pp. 213-222).

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn²⁺ binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 Å that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399–2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.

Keywords: Abbreviations; BoNT; botulinum neurotoxin; LC; light chain; SNAP-25; synaptosomal associated protein of 25 kDa; VAMP; vesicle-associated membrane protein; SNARE; soluble NSF attachment protein receptor; NaPB; sodium phosphate buffer; CD; circular dichroism; PCR; polymerase chain reaction; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisBotulinum neurotoxin; Endopeptidase; Site-directed mutagenesis; SNAP-25; Light chain

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4 by Nao Torimoto; Itsuko Ishii; Masayuki Hata; Yukari Kobayashi; Hiroyoshi Nakamura; Noritaka Ariyoshi; Mitsukazu Kitada (pp. 223-232).

CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than α-hydroxytriazolam at concentrations of more than 60 μM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the α-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both α- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited α-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and α-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe–O–C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.

Keywords: Abbreviations; CYP; cytochrome P450; DHEA; dehydroepiandrosteroneCYP3A4; Triazolam; Endogenous steroid; Theoretical calculation

Compensatory secondary structure alterations in protein glycation by Ranjita GhoshMoulick; Jaydeep Bhattacharya; Shibsekhar Roy; Soumen Basak; Anjan Kr. Dasgupta (pp. 233-242).

Glycation, a local covalent interaction, leads to alterations in secondary and tertiary structures of hemoglobin, the changes produced by fructose being more pronounced than those caused by glucose. The Stokes diameter of hemoglobin increases upon glycation from 7 to 14 nm and a concurrent inter-chain cross-linking and heme loss are also observed, particularly in the later stage of glycation. An initial increase of tryptophan (trp) fluorescence was observed in both glucation and fructation. In case of frucation however there was a decrease in tryptophan fluorescence that was accompanied by an increase in fluorescence of the advanced glycosylation end products (AGEs). This fluorescence behavior is indicative of energy transfer between tryptophan and the AGEs formed during the late stage of glycation. Emergence of an isosbestic point in the fluorescence spectra (taken at different time intervals) implies existence of two distinct glycation stages. The late glycation stage is also marked by an increase of beta structure and random coil at the expense of alpha helix. It is further observed that this compensatory loss of alpha helix (reported for the first time) and increase in beta sheet and random coil elements depend on the number of solvent-accessible glycation sites (rather than total number of such sites) and the subunit assembly of the protein.

Keywords: Glycation; Cross-Linking; Fluorescence; Circular Dichroism; Alpha helix; Beta sheet

Tyrosine83 is essential for the activity of E. coli galactoside transacetylase by A. Radeghieri; M. Bonoli; F. Parmeggiani; A. Hochkoeppler (pp. 243-248).

The gene ( lacA) coding for Escherichia coli galactoside transacetylase was cloned into the pTrcHisB plasmid, and the corresponding hexahistidine-tagged enzyme was over-expressed and purified. The kinetic constants of the tagged protein were determined, yielding values in excellent agreement with previous observations reported for the natural enzyme. LacA Tyrosine83 was then substituted with a Valine: by comparing the K_m and k_cat values observed for wild type and mutant enzymes using isopropyl-thio-β-d-galactopyranoside or p-nitrophenyl-β-d-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.

Keywords: Escherichia coli; Galactoside transacetylase; LacA; Tyrosine83

How does dextran sulfate prevent heat induced aggregation of protein?: The mechanism and its limitation as aggregation inhibitor by Kwanghun Chung; Juhan Kim; Byung-Kwan Cho; Byoung-Joon Ko; Bum-Yeol Hwang; Byung-Gee Kim (pp. 249-257).

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA–dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.

Keywords: Protein aggregation; Dextran sulfate; Demixing; Protein denaturation

Formation of high-molecular-weight protein adducts by methyl docosahexaenoate peroxidation products by Wei Liu; Hua-Jie Wang; Li-Ping Wang; Shan-Lin Liu; Jin-Ye Wang (pp. 258-266).

In the present study, the formation of modified proteins by methyl docosahexaenoate (DHA) peroxidation products in the presence of a metal-catalyzed oxidation system was investigated. Metal-catalyzed oxidation of mixtures containing bovine serum albumin (BSA) and DHA led to formation of two high molecular weight derivatives of BSA. One had a mass of 71.5 kDa as determined by two-dimensional electrophoresis, matrix assisted laser desorption and ionization mass spectrometer (MALDI MS) analysis. The other was estimated to be 93 kDa by SDS-PAGE electrophoresis. The exposure of BSA to DHA also led to the generation of carbonyl groups. Oxygen radical scavengers could inhibit these modifications induced by DHA peroxidation. Furthermore, there was little difference of the peptides mass fingerprinting between the two kinds of modified high-molecular-weight proteins. These results suggest that oxygen radicals formed during lipid peroxidation are involved in the formation of protein derivatives. Our study may be important in the understanding the specific role of docosahexaenoic acid in the formation of modified proteins during aging and its related diseases.

Keywords: DHA peroxidation; Protein modification; Two-dimensional electrophoresis; Western blot analysis; ESR analysis; MALDI MS

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over by Kathleen Deiteren; Georgiana Surpateanu; Kambiz Gilany; Johan L. Willemse; Dirk F. Hendriks; Koen Augustyns; Yves Laroche; Simon Scharpé; Anne-Marie Lambeir (pp. 267-277).

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K_m and k_cat, of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa–Arg substrates. CPM had the highest catalytic efficiency ( k_cat/ K_m) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k_cat and K_m of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.

Keywords: Abbreviations; Bz; benzoyl; CPMwt; CPM wild type; CP(s); carboxypeptidase(s); CV; column volumes; ds; dansyl; FA; furylacryloyl; GEMSA; 2-guanidinoethylmercaptosuccinic acid; GPI; glycosylphosphatidylinositol; hCPM; CPM purified from human prostasomes; MERGETPA; dl; -2-mercaptomethyl-3-guanidoethylthiopropanoic acid; PEP; phosphoenolpyruvate; PI-PLC; phosphatidylinositol-specific phospholipase CCarboxypeptidase; Structure function relationship; Membrane protein; Substrate specificity; Molecular modelling; Peptide

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers by Karina M. Monteiro; Sandra M.N. Scapin; Marcos V.A.S. Navarro; Nilson I.T. Zanchin; Mateus B. Cardoso; Nádya P. da Silveira; Paulo F.B. Gonçalves; Hubert K. Stassen; Arnaldo Zaha; Henrique B. Ferreira (pp. 278-285).

Echinococcus granulosus antigen B is an oligomeric protein of 120–160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed ~120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.

Keywords: Abbreviations; AgB; antigen B; AgB8; 8-kDa AgB subunit; CD; circular dichroism; CHD; cystic hydatid disease; DLS; dynamic light scattering; HF; hydatid fluid Echinococcus granulosus; Antigen B; Recombinant subunits; Oligomerization; Thermostability; Protease sensitivity

Glutamate 2,3-aminomutase: A new member of the radical SAM superfamily of enzymes by Frank J. Ruzicka; Perry A. Frey (pp. 286-296).

A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity towardl-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence ofl-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motifCSMYCRHC corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the β-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.

Keywords: Abbreviations; PLP; pyridoxal-5′-phosphate; SAM; S; -adenosyl-; l; -methionine; EPPS; N; -[2-hydroxyethyl-piperazine-; N; ′-(3-propanesulfonic acid)]; dNTP; a 2′-deoxyribonucleotide-5′-triphosphate; DTT; dithiothreitol; EDTA; ethylenediamine tetraacetic acid; EPR; electron paramagnetic resonance; HPLC; high performance liquid chromatography; IPTG; isopropyl-β-; d; -thiogalactopyranoside; PCR; polymerase chain reaction; PITC; phenylisothiocyanate; PMSF; phenylmethanesulfonyl fluoride; Tris; tris(hydroxymethyl)aminomethane; ENDOR; electron nuclear double resonance; ESEEM; electron spin echo envelope modulation; XAS; X-ray absorption spectroscopy; SDS PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisGlutamate 2,3-aminomutase; Gene for glutamate 2,3-aminomutase; Expression in; E. coli; Characterization of glutamate 2,3-aminomutase; Radical intermediate in glutamate 2,3-aminomutase; New radical SAM enzyme

Basis for the equilibrium constant in the interconversion ofl-lysine andl-β-lysine by lysine 2,3-Aminomutase by Dawei Chen; Justinn Tanem; Perry A. Frey (pp. 297-302).

l-β-lysine and β-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The p K_a values have been titrimetrically measured and are forl-β-lysine: p K₁=3.25 (carboxyl), p K₂=9.30 (β-aminium), and p K₃=10.5 (ε-aminium). For β-glutamate the values are p K₁=3.13 (carboxyl), p K₂=3.73 (carboxyl), and p K₃=10.1 (β-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the β-isomers. The pH and temperature dependencies of K_eq have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of β-lysine. The value of K_eq (8.5 at 37 °C) is independent of pH between pH 6 and pH 11; ruling out differences in p K-values as the basis for the equilibrium constant. The K_eq-value is temperature-dependent and ranges from 10.9 at 4 °C to 6.8 at 65 °C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with ΔH°=−1.4 kcal mol−¹ and ΔS°=−0.25 cal deg−¹ mol−¹. Exothermicity is attributed to the greater strength of the bond C_β—N_β inl-β-lysine than C_α—N_α inl-lysine, and this should hold for other amino acids.

Keywords: Lysine 2,3-aminomutase; β-lysine; p; K; _a; -values; β-glutamate; p; K; _a; -values; K; _eq; of lysine/β-lysine; ΔH° of lysine/β-lysine; ΔS° of lysine/β-lysine

Rv3389C from Mycobacterium tuberculosis, a member of the ( R)-specific hydratase/dehydratase family by Emmanuelle Sacco; Virginie Legendre; Françoise Laval; Didier Zerbib; Henri Montrozier; Nathalie Eynard; Christophe Guilhot; Mamadou Daffé; Annaïk Quémard (pp. 303-311).

The ( R)-specific 3-hydroxyacyl dehydratases/ trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C₈–C₁₆) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.

Keywords: Abbreviations; ACP; acyl carrier protein; Ct; MFE-2; MFE-2 from; Candida tropicalis; DTT; dithiothreitol; Ec; FabA; Ec; FabZ, FabA, FabZ from; E. coli; FAS; fatty acid synthase; FAS-I; type I FAS; FAS-II; type II FAS; hyg; hygromycin; H-Rv3389c; His-tagged Rv3389c protein; MFE-2; multifunctional enzyme type 2; MFE-2hd; hydratase domain of MFE-2; PDB; protein data bank; PHA; polyhydroxyalkanoate; 3; R; HDC; (3; R; )-hydroxydecanoyl-CoA(; R; )-specific 3-hydroxyacyl dehydratase; Enoyl-CoA hydratase; Mycobacterium; Fatty acid biosynthesis; Hydratase 2; Double hot dog fold

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site by Stefano Mangani; Manuela Benvenuti; Arthur J.G. Moir; Maria Ranieri-Raggi; Daniela Martini; Antonietta R.M. Sabbatini; Antonio Raggi (pp. 312-322).

XAS of Zn–peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48–61 of the enzyme contains a zinc binding site, whilst the region 360–372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn–Zn distance of about 3.3 Å. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.

Keywords: Abbreviations; AMPD; AMP deaminase; HPRG; histidine-proline-rich-glycoprotein; XAS; X-ray absorption spectroscopy; EXAFS; Extended X-ray Absorption Fine Structure; FT; Fourier Transforms; BVS; bond valence sum; CD; Circular Dichroism; SOD1; superoxide dismutaseAMP deaminase; Zinc binding site; Histidine-proline-rich-glycoprotein; X-ray absorption spectroscopy

Aggregation as the basis for complex behaviour of cutinase in different denaturants by Daniel E. Otzen; Lise Giehm; Ricardo P. Baptista; Søren R. Kristensen; Eduardo P. Melo; Shona Pedersen (pp. 323-333).

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.

Keywords: Abbreviations; ANS; 8-anilino-1-naphthalenesulphonic acid; DLS; Dynamic light scatteringCutinase; Folding scheme; Denaturant; Guanidinium thiocyanate; Aggregation

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BBA - Proteins and Proteomics (v.1774, #2)