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BBA - Proteins and Proteomics (v.1804, #6)

Editorial Board (pp. i).

Understanding protein non-folding by Vladimir N. Uversky; A. Keith Dunker (pp. 1231-1264).

This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of a specific 3-D structure. The existence of such proteins does not fit the prevailing structure–function paradigm, which states that a unique 3-D structure is a prerequisite to function. Thus, the protein structure–function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: how were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases?

Keywords: Intrinsically disordered protein; Disorder prediction; Partially folded protein

Standardized evaluation of protein stability by Annick Thomas; Bernard Joris; Robert Brasseur (pp. 1265-1271).

We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200–250 residues have equivalent MFP values that are averaged to –65±3kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from –25 for Gly to –115kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of −63.5±2.5kcal/aa, i.e., only 1.5kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.

Keywords: Protein stability; MFP; Standard value; Amyloid fibril; Amino acids; Peptides

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme by Kuniko Akama; Tomoe Horikoshi; Atsushi Sugiyama; Satoko Nakahata; Aoi Akitsu; Nobuyoshi Niwa; Atsushi Intoh; Yasutaka Kakui; Michiko Sugaya; Kazuo Takei; Noriaki Imaizumi; Takaya Sato; Rena Matsumoto; Hitoshi Iwahashi; Shin-ichi Kashiwabara; Tadashi Baba; Megumi Nakamura; Tosifusa Toda (pp. 1272-1284).

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

Keywords: Abbreviations; PDI-P5; protein disulfide isomerase P5; PDIA3; protein disulfide isomerase A3; PBS; phosphate-buffered saline; DTT; dithiothreitol; IPG; immobilized pH gradient; IEF; isoelectric focusing; BPB; Bromophenol Blue; 2-DE; two-dimensional gel electrophoresis; MS; mass spectrometry; T-TBS; TBS containing 0.05% Tween; mPDI-P5; mature protein disulfide isomerase P5; m PDIA3; mature protein disulfide isomerase A3; IPTG; isopropylthiogalactosideProteomics; Two-dimensional gel electrophoresis; Protein disulfide isomerase-P5; Protein disulfide isomerase A3; Sperm maturation; Anti-chaperone activity

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners by Hyun-Hee Jang; Arvind P. Jamakhandi; Shane Z. Sullivan; Chul-Ho Yun; Paul F. Hollenberg; Grover P. Miller (pp. 1285-1293).

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.

Keywords: Abbreviations; CYP2B1; cytochrome P450 2B1; CPR; cytochrome P450 reductase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; cyt; c; cytochrome; c; (horse heart); cyt; b; ₅; cytochrome; b; ₅; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 7-ethoxy-4-trifluoromethylcoumarin; 7-EFC; 7-hydroxy-4-trifluoromethylcoumarin; 7-HFC; NADP; ⁺; nicotinamide adenosine dinucleotide phosphate (oxidized); I; ionic strengthCytochrome P450 reductase; Docking; Site-directed mutagenesis; CYP2B1; Protein–protein interactions; Electrostatic interactions

Alkaline pH-dependent differential unfolding characteristics of mesophilic and thermophilic homologs of dimeric serine hydroxymethyltransferase by Anant Narayan Bhatt; Vinod Bhakuni; Ashutosh Kumar; M. Yahiya Khan; Mohammad Imran Siddiqi (pp. 1294-1300).

Environmental variables such as pH can significantly influence the folding and stability of a protein molecule. In the present investigation, we compared the alkaline pH-induced unfolding of two homologous serine hydroxymethyltransferase from mesophilic Bacillus subtilis (bsSHMT) and thermophilic Bacillus stearothermophilus (bstSHMT) using various biophysical techniques. The thermophilic enzyme bstSHMT was found to be more resistant to alkaline denaturation compared to its mesophilic counterpart, bsSHMT. Unfolding studies using domain-swapped chimera, constructed by swapping the C-terminal domain of these two wild-type proteins, revealed that C-terminal domain plays a pivotal role in the folding, stability and subunit interaction of these proteins. Primary amino acid sequence analysis of the proteins showed that bsSHMT has six unconserved lysine residues in C-terminal domain, which are absent in bstSHMT. Chemical modification of lysine side chains resulted in stabilization of monomers, only in case of bsSHMT. Moreover, comparison between homology model of bsSHMT with the crystal structure of bstSHMT revealed that a small stretch of 11 amino acids at the end of C-terminal domain was found protruding outside the molecule as a flexible coiled structure in bsSHMT. Taken together these findings suggest that possibly the presence of these non-identical lysine moieties and a small extension of C-terminal domain may be responsible for low stability of bsSHMT under alkaline pH condition.

Keywords: Abbreviations; SHMT; Serine hydroxymethyltransferase; PLP; Pyridoxal-5′-phosphate; SEC; Size-exclusion chromatography; CD; Circular Dichroism; CGH; Citrate Glycine Hepes bufferAcetylation; Alkaline denaturation; Chimera; Homologous proteins

DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis by Haiming Li; Limei Chang; Jenika M. Howell; Raymond J. Turner (pp. 1301-1309).

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

Keywords: Chaperones; Molybdenum cofactor biosynthesis pathway; DmsD; Protein–protein interactions; Tat

Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions by Yu-Chiang Hung; Pei-Wen Wang; Tai-Long Pan (pp. 1310-1321).

Salvia miltiorrhiza is a Chinese herb widely used for cardiovascular disorder regimens, yet little is known about the cellular mechanisms that contribute to attenuated growth of smooth muscle cells (SMCs) under oxidative stress such as homocysteine (Hcy) treatment. As anticipated, a low dose (0.015 mg/mL) of S . miltiorrhiza aqueous extract (SMAE) significantly inhibited (>60%) the growth of a rat smooth muscle cell line (A10) under Hcy stimulation and the intracellular reactive oxygen species (ROS) concentration obviously decreased after SMAE treatment in terms of reducing p47^phox translocation and increasing catalase activity. Signaling profile suggests that SMAE inhibited Hcy-induced A10 cell growth via the PKC/MAPK-dependent pathway. Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 14 proteins in response to Hcy and Hcy/SMAE. Meanwhile, SMAE attenuated carbonyl-modification of specific cytoskeleton and chaperone proteins leading to cell type transformation. Moreover, a network analysis using MetaCore™ shed more light on the molecular basis associated with SMAE efficacy. SMAE exerts its protective effect through the scavenging of ROS and subsequent modulation of protein carbonylation to inhibit cell proliferation. These signature networks and functional proteomics highlighted herein may facilitate the evaluation of potential therapeutic targets and elucidate novel mechanisms through which protein functions can be regulated by the redox status.

Keywords: Salvia miltiorrhiza; Homocysteine; A10 cells; Two-dimensional electrophoresis; Mass spectrometry; Carbonylation; Reactive oxygen species; Network analysis

Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity by Rene-Trouillefou Malika René-Trouillefou; Amal Benzaria; Stéphane Marchal; Reinhard Lange; Bertrand Caporiccio; Eliane Dumay (pp. 1322-1333).

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600MPa and 45°C in Tris–HCl buffer (20mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20°C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8M urea at atmospheric pressure or at 600MPa and 20°C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45°C only. High pressure up to 600MPa induced spectral changes which at 20°C were fully reversible upon decompression. At 45°C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8M further increased SEA structural changes at 600MPa and 20°C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.

Keywords: Isostatic high pressure; Enterotoxin A; Staphylococcus aureus; (SEA); Fluorescence; T-cell; Thymocyte proliferation

Mass spectrometry analysis of complexes formed by myotonic dystrophy protein kinase (DMPK) by Francesca Forner; Sandra Furlan; Sergio Salvatori (pp. 1334-1341).

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats at the 3′-UTR of the serine/threonine protein kinase DMPK. Expanded CTG repeats are toxic since they are transcribed into an RNA molecule which is then sequestered within the nucleus in the form of foci. RNA cytotoxicity is linked to the aberrant splicing of several developmentally regulated genes. DMPK transcripts undergo alternative splicing giving rise to many isoforms but do not seem to be involved in the splicing dysregulation of DM1. However, decreased levels of DMPK in DM1 patients and DMPK involvement in muscle weakness and cardiac dysfunction in animal models have been reported. The variability in phenotypic expression of DMPK together with its differential subcellular targeting, suggests that different splicing isoforms may be involved in different signalling pathways, possibly through DMPK-interacting proteins. To gain better insight into the DMPK function, we used mass spectrometry to identify proteins co-segregating with DMPK in soluble complexes isolated from high-speed supernatant of rat muscles. We carried out experiments with native DMPK to preserve the physiological stoichiometry with potential partners. DMPK-containing complexes were isolated and immuno-detected by non-denaturing electrophoresis, gel filtration, ionic-exchange chromatography and immunoprecipitation. DMPK peptides were identified by high-resolution mass spectrometry together with several putative DMPK-binding proteins, including several heat shock proteins such as HSP20/HSPB6, HSP60/CPN60, HSP70 and HSP90. We also obtained evidence of a direct interaction of DMPK with αB-crystallin/HSPB5 and HSP25/HSPB1.

Keywords: DMPK; Protein–protein interaction; Mass spectrometry; Non-denaturing electrophoresis; Co-immunoprecipitation; αB-crystallin/HSPB5; HSP25/HSPB1

Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM) by Mariana Figuera-Losada; Marie Ranson; Martina L. Sanderson-Smith; Mark J. Walker; Francis J. Castellino; Mary Prorok (pp. 1342-1349).

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding groupA streptococcalM-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand–protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients ( S°_20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.

Keywords: Abbreviations; 6-AHA; 6-aminohexanoic acid; GAS; group A streptococcus; PAM; plasminogen binding group A streptococcal M-like protein; Pg; plasminogen; Pm; plasmin; SK; streptokinase; uPA; urokinase-type Pg activatorPlasminogen; Streptokinase; Group A streptococci; M-like protein; PAM

Unraveling an FNR based regulatory circuit in Paracoccus denitrificans using a proteomics-based approach by Pavel Bouchal; Struharova Iva Struhárová; Budinska Eva Budinská; Ondrej Šedo; Vyhlidalova Tereza Vyhlídalová; Zdrahal Zbyněk Zdráhal; Rob van Spanning; Igor Kučera (pp. 1350-1358).

The switch from aerobic to anaerobic respiration in the bacterium Paracoccus denitrificans is orchestrated by the action of three FNR-type transcription regulators FnrP, NNR and NarR, which are sensors for oxygen, nitric oxide and nitrite, respectively. In this work, we analyzed the protein composition of four strains (wild type, FnrP-, NNR- and NarR-mutant strains) grown aerobically, semiaerobically and semiaerobically in the presence of nitrate to discover the global role of FNR-family transcription regulators using proteomics, with data validation at the transcript and genome levels. Expression profiles were acquired using two-dimensional gel electrophoresis for 737 protein spots, in which 640 proteins were identified using mass spectrometry. The annotated 2-D proteome map provided the most comprehensive coverage of P. denitrificans proteome available to-date and can be accessed on-line athttp://www.mpiib-berlin.mpg.de/2D-PAGE/. Our results revealed several types of regulation under the conditions tested: (1) FnrP-controlled regulation of nitrous oxide reductase, UspA and OmpW as confirmed at protein, transcript and DNA level (position of FNR boxes). (2) Proteins regulated via additional regulators, including proteins involved in NNR and NarR regulons: nitrate reductase β-subunit, TonB-dependent receptors, nitrite reductase, a TenA-type transcription regulator, and an unknown protein with an alpha/beta hydrolase fold. (3) Proteins whose expression was affected mainly by the growth condition. This group contains SSU ribosomal protein S305 / σ⁵⁴ modulation protein, and two short-chain reductase–dehydrogenase proteins.

Keywords: Paracoccus denitrificans; FNR transcription regulator; Respiratory switch; Two-dimensional gel electrophoresis; Proteome

X-ray structures of Bacillus pallidusd-arabinose isomerase and its complex withl-fucitol by Kosei Takeda; Hiromi Yoshida; Ken Izumori; Shigehiro Kamitori (pp. 1359-1368).

d-Arabinose isomerase (d-AI), also known asl-fucose isomerase (l-FI), catalyzes the aldose–ketose isomerization ofd-arabinose tod-ribulose, andl-fucose tol-fuculose. Bacillus pallidus ( B. pallidus)d-AI can catalyze isomerization ofd-altrose tod-psicose, as well asd-arabinose andl-fucose. Three X-ray structures of B. pallidusd-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor,l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60Å, respectively. B. pallidusd-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (β/α) barrel fold domain. A catalytic metal ion (Mn²⁺) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose–ketose isomerization by B. pallidusd-AI, as well as Escherichia coli ( E. coli)l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidusd-AI possibly interconverts between “open” and “closed” forms, and that the additional metal ion found in B. pallidusd-AI may help to stabilize the channel region.

Keywords: Abbreviations; d; -AI; d; -arabinose isomerase; E. coli; Escherichia coli; l; -FI; l; -fucose isomerase; l; -AI; l; -arabinose isomerase; B. pallidus; Bacillus pallidusX-ray structure; d; -Arabinose isomerase; l; -Fucose isomerase; Bacillus pallidus; Enzyme/substrate complex

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase by Vinayakumar Gedi; Kumaresan Jayaraman; Satish Kalme; Hye-Yeon Park; Hae-Chul Park; Im-Joung La; Hoh-Gyu Hahn; Moon-Young Yoon (pp. 1369-1375).

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC₅₀ values of 1.0±0.02, 1.0±0.04, 2.1±0.12, and 2.0±0.08µM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC₅₀ values of 0.05±0.002, 0.153±0.004, 1.30±0.10, and 1.29±0.40µM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1 H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, −8.52kcal/mol and formed a single hydrogen bond with a distance of 1.973Ǻ. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.

Keywords: Abbreviations; Bantx; B. anthracis; HE; reconstituted AHAS; BCAAs; branched-chain amino acids; CSU; catalytic subunit; RSU; regulatory subunit; SUs; sulfonylureas; IMs; imidazolinones; TPs; triazolopyrimidinesAcetohydroxyacid synthase; Bacillus anthracis; Docking; Reconstitution; Triazol-1-yl-pyrimidines

Differential modulation of the active site environment of human carbonic anhydrase XII by cationic quantum dots and polylysine by Sumathra Manokaran; Xing Zhang; Wei Chen; D.K. Srivastava (pp. 1376-1384).

Due to prevalence of negative charges on the protein surface, opposite to the active site pocket of human carbonic anhydrase XII (hCA XII), both positively charged CdTe quantum dots (Qds⁺) and polylysine electrostatically interact with the enzyme, and such interaction does not influence the catalytic activity of the enzyme. However, both these cationic macromolecules differently modulate the active site environment of the enzyme. The steady-state kinetic data revealed that whereas polylysine exhibited no influence on dansylamide (DNSA) dependent inhibition of the enzyme, Qds⁺ overcame such an inhibitory effect, leading to almost 70% restoration of the catalytic activity of the enzyme. We provide evidence that DNSA remains bound to the enzyme upon interaction with both polylysine and Qds⁺. Arguments are presented that the above differential feature of polylysine and Qds⁺ on hCA XII is encoded in the “rigidity” versus “flexibility” of these cationic macromolecules.

Keywords: Abbreviations; CA; carbonic anhydrase; hCA XII; human carbonic anhydrase XII; DNSA; dansylamide; CdTe; cadmium telluride; Qds; CdTe quantum dots; Qds; ⁺; positively charged quantum dots; EDTA; Ethylene diamine tetra acetic acid; PMSF; phenylmethylsulfonyl fluoride; IPTG; Isopropyl-beta-; d; -thiogalactopyranosideCarbonic anhydrase XII; Quantum dot; Dansylamide; Polylysine

Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils by Edita Gaidamaviciute; Daiva Tauraite; Julius Gagilas; Arunas Lagunavicius (pp. 1385-1393).

We present site-directed chemical modification (SDCM), a tool for engineering U-resistant archaeal DNA polymerases of family B. The Thermococcus litoralis Sh1B DNA polymerase (GenBank: GQ891548) was chosen as the object of the study. Similar to D.Tok, Kod1, Pfu, Tgo and other archaeal members of this family, the T. litoralis Sh1B DNA polymerase is a domain structured, proofreading-proficient enzyme that has the polymerization and 3′→5′ DNA exonucleolytic activities and contains N-terminally located highly conserved template-strand U-binding pocket. The tight binding of template uracil in the enzyme pocket during polymerization blocks the replication of DNA containing uracils. This effect can be alleviated by mutations in key amino acids of the U-binding pocket. We altered T. litoralis Sh1B DNA polymerase's ability to read through the template-strand uracils by applying SDCM. Specific modification of individual cysteine residues in U-binding pocket — targets introduced into certain positions by site-directed mutagenesis — enables the enzyme to effectively replicate DNA containing uracils. We demonstrate that the acquired resistance of chemically modified T. litoralis Sh1B DNA polymerase to DNA uracil correlates with its decreased affinity for template-strand uracil.

Keywords: Abbreviations; EMSA; electrophoretic mobility shift assay; SDCM; site-directed chemical modification; T4 PNK; T4 polynucleotide kinaseArchaeal DNA polymerase; Read-ahead; Chemical modification; Thermococcus litoralis; U-binding pocket

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G by Moo-Jin Suh; David J. Clark; Prashanth P. Parmer; Robert D. Fleischmann; Scott N. Peterson; Rembert Pieper (pp. 1394-1404).

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.

Keywords: Abbreviations; PTMs; post-translational modifications; PMF; peptide mass fingerprinting; TMPP-Ac-OSu; N; -succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide; SasG; Staphylococcus aureus; surface protein G; ESI-MS; electrospray ionization mass spectrometry; MALDI-TOF; matrix-assisted laser desorption ionization-time of flight; TFA; trifluoroacetic acid; 2DE; two dimensional gel electrophoresis; CID; collision-induced dissociation; PBS; phosphate buffered saline; EDTA; ethylenediaminetetraacetic acid; BAM; benzamidine-HCl; PMSF; phenylmethylsulphonyl fluorideKeyword; N-terminal truncation; TMPP labeling; Multiple enzyme digestion; SasG; Mass spectrometry; Post-translational modifications

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BBA - Proteins and Proteomics (v.1804, #6)