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BBA - Proteins and Proteomics (v.1764, #11)

Editorial Board (pp. ii).

Modeling electrostatic effects in proteins by Arieh Warshel; Pankaz K. Sharma; Mitsunori Kato; William W. Parson (pp. 1647-1676).

Electrostatic energies provide what is perhaps the most effective tool for structure–function correlation of biological molecules. This review considers the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. We focus on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric ‘constants’ in semimacroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering a wide range of functional properties including p K_a's, redox potentials, ion and proton channels, enzyme catalysis, ligand binding and protein stability. We conclude by pointing out that, despite the current problems and the significant misunderstandings in the field, there is an overall progress that should lead eventually to quantitative descriptions of electrostatic effects in proteins and thus to quantitative descriptions of the function of proteins.

Keywords: Electrostatic effect; Enzyme catalysis; Ion channel; Proton channel; Generalized Born model; Macroscopic model; PDLD/S-LRA; Convergence; Long-range effect; Helix dipole; COmputer modeling; p; K; a

Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs by Tsutomu Arakawa; Daisuke Ejima; Yoshiko Kita; Kouhei Tsumoto (pp. 1677-1687).

A great deal of attention has been paid to so-called amyloid diseases, in which the proteins responsible for the cell death and resultant diseases undergo conformational changes and aggregate in vivo, although whether aggregate formation is the cause or the result of the cell death is controversial. Recently, an increasing attention is given to protein folding diseases tightly associated with mutations. These mutations result in temperature-dependent misfolding and hence inactivation of the proteins, leading to loss of function, at physiological temperature; at low so-called permissive temperatures, the mutant proteins correctly fold and acquire functional structure. Alternatively, activation can be induced by use of osmolytes, which restores the folding of the mutant proteins and hence are called chemical chaperones. The osmolytes are compatible with macromolecular function and do stabilize the native protein structure. However, chemical chaperones require high concentrations for effective folding of mutant proteins and hence are too toxic in in-vivo applications. This limitation can be overcome by pharmacological chaperones, whose functions are similar to the chemical chaperones, but occur at much lower concentrations, i.e., physiologically acceptable concentrations. Although the research and clinical importance of pharmacological chaperones has been emphasized, the initial and central concept of osmolytes is largely ignored. Here we attempt to bridge the concept of osmolytes to applications of pharmacological chaperones.

Keywords: Abbreviations; CFTR; cystic fibrosis transmembrane conductance regulator; ER; endoplasmic reticulum; TMAO; trimethylamine N-oxide; DMSO; dimethyl sulfoxidePharmacological chaperone; Chemical chaperone; Osmolyte; Stabilization

Binding model of human coactosin-like protein with filament actin revealed by mutagenesis by Haiming Dai; Wei Huang; Jian Xu; Bo Yao; Shangmin Xiong; Husheng Ding; Yajun Tang; Haiyan Liu; Jihui Wu; Yunyu Shi (pp. 1688-1700).

Human coactosin-like protein (CLP) is a small (MW ∼17 kDa) evolutionarily conserved actin-binding protein. It can bind to actin filaments but not globular actin and belongs to the fourth class of ADF-H-domain-containing proteins. Human CLP can also bind to 5LO, which plays an important role in cellular leukotriene synthesis. Although the structure of hCLP has been determined by both NMR and X-ray experiments, how hCLP binds to the actin filament is still a controversial question. To obtain insights into the structure of the complex, we studied the three-dimensional structure and backbone dynamics of hCLP using multidimensional NMR spectroscopy. Guided by the solution structure of the protein, a series of site-directed mutants were generated and their F-actin-binding activities were measured by high-speed cosedimentation assays. Furthermore, the structure model of the hCLP-F-actin complex was proposed using computational docking with the docking results filtered by the mutation data. Several previously untested residues (including T66, L89, R91, K102, D116 and E119) in hCLP were found important for the F-actin-binding activity. The extended region of β4–β5 of hCLP (residue 66–75) was found very flexible and very important for F-actin binding. The C-terminal residues of hCLP were not involved in F-actin binding, which was different from UNC-60B. Based on our hCLP-F-actin-binding model, different affinities of the four classes of ADF-H domain containing proteins for F-actin were explained.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; hCLP; human coactosin-like protein; ADF; actin depolymerization factor; ADF-H; actin depolymerization factor homology; 5LO; 5-lipoxygenase; CSI; chemical shift index; RMSD; root–mean–square deviation; T; ₂; transverse relaxation time; T; ₁; longitudinal relaxation time; S; ²; Squared order parameter; τ; _e; the effective correlation time for fast internal motions; K; _d; dissociation constantHCLP; Actin; NMR; Complex model

Bacterially expressed and optimized recombinant Phl p 1 is immunobiochemically equivalent to natural Phl p 1 by Roland Suck; Timo Kamionka; Brigitte Schäffer; Rüdiger Wahl; Andreas Nandy; Bernhard Weber; Arnd Petersen; Ruth Birner-Grünberger; Siva Charan D.V.; Walter Keller; Helmut Fiebig; Oliver Cromwell (pp. 1701-1709).

Recombinant production in bacteria of soluble and monomeric Phl p 1, a major allergen of Timothy grass pollen, has proved to be very problematic. In order to facilitate expression and purification of this allergen, a recombinant variant was designed with a single amino acid substitution. Several comparative analyses with natural counterparts using electrophoretic and HPLC separations, together with immunological assays, demonstrated high equivalence. This is the first description of an approach aiming at an improvement of a natural like recombinant allergen.

Keywords: rPhl p 1 variant; Dac g 1; Lol p 1; Bacterial expression; IgE-binding; Peptide mapping

Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different by Irina P. Gileva; Tatiana S. Nepomnyashchikh; Denis V. Antonets; Leonid R. Lebedev; Galina V. Kochneva; Antonina V. Grazhdantseva; Sergei N. Shchelkunov (pp. 1710-1718).

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand–receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.

Keywords: Orthopoxvirus; TNF-binding protein; Baculovirus expression; Septic shock

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA by Ping Xie (pp. 1719-1729).

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.

Keywords: Helicase; Translocation mechanism; Unwinding mechanism; Molecular motor

The interaction between plasminogen and antiplasmin variants as studied by surface plasmon resonance by Haiyao Wang; Anna Karlsson; Iréne Sjöström; Björn Wiman (pp. 1730-1734).

The interaction between immobilized plasminogen or an elastase-degradation product from plasminogen, constituting “kringles�? 1–3 and different purified variants of antiplasmin has been studied by surface plasmon resonance utilising a BIAcore. The antiplasmin variants studied are wild-type, K429E, K436E, E443G, D444G, K452E and K452T. It is shown that the two mutants K452T and K452E react in quite a similar way as wt-antiplasmin, suggesting that Lys452 is not involved in the lysine-binding site interaction between plasminogen and antiplasmin. On the other hand, the mutant K436E displays a much lower k_a. The affinity between plasminogen or the fragment constituting “kringles�? 1–3 and K436E were also much lower than with wt-antiplasmin. Thus, also the data obtained with surface plasmon resonance show that Lys436 indeed is very important in the lysine-binding site mediated interaction between plasminogen and antiplasmin.

Keywords: Antiplasm; Plasminogen; Surface plasmon resonance

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues by Johanna L. Hellström; Markus Vehniäinen; Merja Mustonen; Timo Lövgren; Urpo Lamminmäki; Jukka Hellman (pp. 1735-1740).

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 °C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 °C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.

Keywords: Abbreviations; PGAP; pyroglutamate aminopeptidase (pyrrolidone carboxyl peptidase); PVDF; polyvinylidene difluoride; DTT; dithiotreitol; PTH; phenylthiohydantoin; Mab; monoclonal antibody; PVP; polyvinyl pyrrolidoneImmunoglobulin; Pyroglutamate aminopeptidase; Unfolding; Deblocking; Aggregation; Protein sequencing

Reversible self-association of recombinant bovine factor B by Grigory I. Belogrudov; Virgil Schirf; Borries Demeler (pp. 1741-1749).

The recombinant bovine factor B, obtained by a newly developed bacterial expression system, was found to exhibit features characteristic of a reversible self-associating system. Using size-sieving chromatography, distribution of the factor B species ranged from a monomer to a trimer, but not oligomers of higher molecular weights. At high protein concentrations, factor B migrated as a single band in a native gel. Cross-linking with the amino-reactive cross-linking reagent bis (sulfosuccinimidyl) suberate (BS), at a low cross-linker to protein ratio yielded cross-linked products identified as factor B dimer and trimer. The cross-linking pattern was shown to be a function of the protein and cross-linker concentrations. The range of sedimentation coefficients in a sedimentation velocity experiment suggested that the largest particle present in the distribution was more than twice as large as the smallest. The data obtained under multiple conditions in the sedimentation equilibrium experiments are best fit to a model describing a reversible self-association of a monomer–trimer of factor B species, with a dissociation constant Kd_1,3=2.48×10−¹⁰ M².

Keywords: Abbreviations; AE-SMP; ¹; SMP depleted of factor B; SMP; bovine heart submitochondrial particles; FB; a regulatory component of ATP synthase complex factor B/subunit; s; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; DTT; dithiothreitol; TCEP; Tris (2-carboxyethylphosphine) hydrochloride; EDTA; ethylenediaminetetraacetic acid; Tris; Tris (hydroxymethyl) aminomethane; IPTG; isopropyl-β-; d; -thiogalactopyranoside; BS; Bis (sulfosuccinimidyl) suberate; sulfo-DST; disulfosuccinimidyl tartrate; EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlorideFactor B; Energy coupling; Proton leak; Cross-link; Dimer; Trimer; Self-association; Analytical ultracentrifugation; Sedimentation equilibrium; Sedimentation velocity; UltraScan

Dendrimer–protein interactions studied by tryptophan room temperature phosphorescence by Edi Gabellieri; Giovanni B. Strambini; Dzmitry Shcharbin; Barbara Klajnert; Maria Bryszewska (pp. 1750-1756).

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.

Keywords: PAMAM dendrimers; Dendrimer–protein interactions; Protein conformation; Room temperature phosphorescence

Human complement factor I glycosylation: Structural and functional characterisation of the N-linked oligosaccharides by Stefanos A. Tsiftsoglou; James N. Arnold; Pietro Roversi; Max D. Crispin; Catherine Radcliffe; Susan M. Lea; Raymond A. Dwek; Pauline M. Rudd; Robert B. Sim (pp. 1757-1766).

Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25–27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A₂G₂S₂, a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH₃) indicating that the charged glycans of fI do not influence the fI-cofactor–substrate interactions.

Keywords: Abbreviations; 2-AB; 2-aminobenzamide; ABS; sialidase from; Arthrobacter urefaciens; for α(2–3) and α(2–6) linked sialic acid; Ac; N; -acetyl; BKF; Fucosidase from bovine kidney; BTG; β-galactosidase from bovine testis; C3(H; ₂; O)/ C4(H; ₂; O); C3/C4 with hydrolysed thioester; C3(NH; ₃; ); A structural analog of C3(H; ₂; O). C3(NH; ₃; ) is formed when the C3 thioester is cleaved by nucleophilic attack by NH; ₃; CV; Column Volume; fB; Complement factor B; fD; Complement factor D; fH; Complement factor H; fI; Complement factor I; FIMAC; Factor I Membrane Attack Complex; FPLC; Fast Performance Liquid Chromatography; GlcNAc; N; -acetylglucosamine; GU; Glucose Unit; GuH; Recombinant β-; N-; acetylglucosaminidase from; Streptococcus pneumonia; HC; Heavy chain of fI; HEPES; 2-[4-(2-hydroxyethyl-1-piperazine)]ethanesulphonic acid; iC3b; Inactivated C3b (cleaved by factor I); LC; Light chain of fI; LDLR-A; Low Density Lipoprotein Receptor type A; NanI; Recombinant sialidase from; Streptococcus pneumonia; preferentially cleaves α(2–3) linked sialic acid; N; ^x; Asparagine with attached carbohydrate; NP-HPLC; Normal Phase High Performance Liquid Chromatography; PBS; Phosphate-Buffered Saline (Dulbecco A formula: 8.2 mM Na; ₂; HPO; ₄; , 1.5 mM KH; ₂; PO; ₄; , 139 mM NaCl, 3 mM KCl, pH 7.4); PNGase F; Peptide-; N; ⁴; -(acetyl-beta-glucosaminyl)-asparagine amidase; SDS-PAGE sample buffer; 0.2 M Tris, 8 M urea, 2% (w/v) SDS, 2 mM EDTA, pH 8.0 with 0.001% (w/v) Bromophenol Blue and 40 mM 1,4-dithiothreitol (reducing conditions) or 20 mM iodoacetamide (alkylating conditions); SDS; Sodium Dodecyl Sulphate; SDS-PAGE; Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SP; Serine Protease; SRCR; Scavenger Receptor Cysteine RichHuman; Complement; Factor I; Serine protease; N; -glycosylation; Sialylation

Do properties of bovine serum albumin at fluid/electrolyte interface follow the Hofmeister series?—An analysis using Langmuir and Langmuir–Blodgett films by Muthuselvi Lakshmanan; R. Parthasarathi; Aruna Dhathathreyan (pp. 1767-1774).

Folding and solubility of proteins are dependent on their state of hydration. How does a protein—bovine serum albumin (BSA) behave in the presence of Hofmeister electrolytes, especially at interfaces? Langmuir films of bovine serum albumin (BSA) in the presence of different Hofmeister electrolytes at air/solution interface and as Langmuir–Blodgett films (LB films) at solid/solution interface have been studied using the surface pressure–molecular area ( π– A) isotherms and surface energy parameters. Changes in secondary structure have been analyzed using circular dichroism (CD) and fluorescence spectroscopy. Hydrodynamically coupled water fraction of BSA in different environments has been estimated using quartz crystal microbalance (QCM) and related to the secondary structural changes. Molecular modeling of BSA in different environments showed that the protein has a compact structure at the interface compared to vacuum. The contact areas estimated using molecular modeling agreed with the experimental results.The results show that the properties of BSA at the interface follow the Hofmeister series with NaF leading to maximum compaction in the protein. Further, in addition to ion specific solvation and different ion size, water structure alteration and the bound water fractions contribute importantly to the Hofmeister effect.

Keywords: BSA; fluid/electrolyte interface; Langmuir; Langmuir–Blodgett film; coupled water

Proteome analysis of human follicular fluid by Stefania Angelucci; Domenico Ciavardelli; Fabrizio Di Giuseppe; Enrica Eleuterio; Marilisa Sulpizio; Gian Mario Tiboni; Franca Giampietro; Paola Palumbo; Carmine Di Ilio (pp. 1775-1785).

We used proteomic approach to analyze the protein profile of human follicular fluid (HFF) obtained from 25 normo-ovulatory women undergoing assisted reproduction techniques due to a male infertility factor. In all HFF samples analyzed we found 695 common spots distributed in the 3 to 10 pH range and in the 10–200 kDa range. Only 625 of these spots were also present in the plasma. We used MALDI-TOF-MS analysis to unequivocally assign 183 HFF/plasma matched spots and 27 HFF/plasma unmatched spots. A large number of acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin and plasma amyloid protein, were identified in HFF in relatively high concentration supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. We also identified several important antioxidant enzymes; i.e., catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27 and protein disulfide isomerase. This indicates that during maturation the human follicle is well protected against toxic injury due to oxidative stress.

Keywords: Abbreviations; APP; acute phase proteins; GST; glutathione; S; -transferase; HFF; human follicular fluid; IGFBP-1; insulin-like growth factor binding protein 1; LDH; l; -lactate dehydrogenase; PP12; placental protein 12; SAP; amyloid plasma P proteinProteome analysis; Human follicular fluid; Folliculogenesis; Acute-phase response protein

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BBA - Proteins and Proteomics (v.1764, #11)