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BBA - Proteins and Proteomics (v.1834, #4)

Editorial Board (pp. i).

Position-specific scoring matrix and hidden Markov model complement each other for the prediction of conopeptide superfamilies by Dominique Koua; Silja Laht; Lauris Kaplinski; Stocklin Reto Stöcklin; Maido Remm; Philippe Favreau; Frédérique Lisacek (pp. 717-724).

Classified into 16 superfamilies, conopeptides are the main component of cone snail venoms that attract growing interest in pharmacology and drug discovery. The conventional approach to assigning a conopeptide to a superfamily is based on a consensus signal peptide of the precursor sequence. While this information is available at the genomic or transcriptomic levels, it is not present in amino acid sequences of mature bioactives generated by proteomic studies. As the number of conopeptide sequences is increasing exponentially with the improvement in sequencing techniques, there is a growing need for automating superfamily elucidation. To face this challenge we have defined distinct models of the signal sequence, propeptide region and mature peptides for each of the superfamilies containing more than 5 members (14 out of 16). These models rely on two robust techniques namely, Position-Specific Scoring Matrices (PSSM, also named generalized profiles) and hidden Markov models (HMM). A total of 50 PSSMs and 47 HMM profiles were generated. We confirm that propeptide and mature regions can be used to efficiently classify conopeptides lacking a signal sequence. Furthermore, the combination of all three-region models demonstrated improvement in the classification rates and results emphasise how PSSM and HMM approaches complement each other for superfamily determination. The 97 models were validated and offer a straightforward method applicable to large sequence datasets.► Successful conopeptide superfamily classification based on pro- and mature peptides ► Combination of HMM and PSSM approaches improved conopeptide superfamily prediction. ► Our method is more efficient and more easily amenable to large data sets than BLAST.

Keywords: Conopeptide; Position specific scoring matrix; Hidden Markov model; Combined prediction; Protein family prediction

Intrinsically disordered regions of p53 family are highly diversified in evolution by Bin Xue; Celeste J. Brown; A. Keith Dunker; Vladimir N. Uversky (pp. 725-738).

Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism's development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.Display Omitted► Evolutionary conservation of the p53 family members is analyzed. ► The ordered DNA-binding domain of p53 is evolutionary conserved. ► p53 disordered regions are characterized by high sequence diversity. ► In evolution, intrinsic disorder and sequence divergence are positively correlated. ► This provides strong support for the existence of disordered regions in p53 in vivo.

Keywords: Abbreviations; ASA; solvent accessible surface area; CDF; cumulative distribution function; CH; charge-hydropathy; CTD; C-terminal domain; DBD; DNA binding domain; ER; endoplasmic reticulum; IDP; intrinsically disordered protein; IDPR; intrinsically disordered protein region; MHC; major histocompatibility complex; MoRFs; molecular recognition features; NORS; no-regular secondary structure; OD; oligomerization domain; PR; proline-rich region; SAM; sterile-α motif; TAD; transactivation domain; UPR; unfolded protein responseIntrinsically disordered proteins; Protein evolution; Protein–protein interactions; Protein–DNA interaction; p53 family

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding by Varhac Rastislav Varhač (pp. 739-744).

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k_a increased 20-fold when the concentration of urea was raised from 0M to 6M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the p K of acid-induced unfolding, the k_a value reached its maximum ( k_a,max) in all urea concentrations. Interestingly, the k_a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.► The association rate constant of CN⁻ binding to heme iron of cyt c is affected by urea. ► The values of k_a,max increase exponentially with increase in urea concentration. ► Urea is a proper modifier of cyt c polypeptide chain flexibility.

Keywords: Conformational flexibility; Cyanide binding; Cytochrome; c; Structural transition; Urea

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase by Kevser Biberoglu; Lawrence M. Schopfer; Ashima Saxena; Ozden Tacal; Oksana Lockridge (pp. 745-753).

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI–TOF–TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.Display Omitted► Polyproline peptides are present in fetal bovine serum acetylcholinesterase. ► We isolated five types of polyproline peptide. ► One type was composed solely of prolines, 13 to 25 residues in length. ► Four types included a single non-proline terminal residue. ► The serum acetylcholinesterase tetramer is organized around a polyproline peptide.

Keywords: Abbreviations; AChE; acetylcholinesterase; BChE; butyrylcholinesterase; MALDI–TOF–TOF; matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry; ColQ; a collagen like protein; PRiMA; proline rich membrane; PRAD; proline-rich attachment domain; FBS; fetal bovine serum; MS; mass spectrometry; MSMS; tandem mass spectrometry; BLAST; basic logical alignment search tool; LC/MSMS; liquid chromatography/mass spectrometry including a peptide fragmentation stage; LTQ-Orbitrap; linear ion-trap quadrupole mass spectrometer; NCBInr; National Center for Biotechnology Information non-redundant databaseFetal bovine serum acetylcholinesterase; Polyproline peptide; Mass spectrometry; PAGE gel electrophoresis; Tetramer organization

Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis by Pesic Ivana Pešić; Muller Gerhard A. Müller; Cosima Baumann; Gry H. Dihazi; Michael J. Koziolek; Marwa Eltoweissy; Carsten Bramlage; Abdul R. Asif; Hassan Dihazi (pp. 754-762).

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5min after beginning, during 5h of treatment, as well as 5min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies.Display Omitted► Intermittent hemodialysis is a form of renal replacement therapy. ► Clinicians prefer to use synthetic dialyzers because of the efficient removal of uremic toxins. ► Our data revealed strong interaction of synthetic dialyzer materials with plasma proteins ► This interaction results in plasma protein removal and membrane occlusion.

Keywords: Abbreviations; ESRD; end-stage renal disease; GFS +; 16; low-flux modified cellulosic membranes dialyzer; GFS +; 20; high-flux modified cellulosic membranes dialyzer; Polyflux 14L; low-flux synthetic membranes dialyzer; Polyflux 140H; high-flux synthetic membranes dialyzer; UFC; ultrafiltration coefficientHemodialysis; Modified cellulose membrane; Synthetic membrane; Proteomics

Unraveling protein stabilization mechanisms: Vitrification and water replacement in a glass transition temperature controlled system by N. Grasmeijer; M. Stankovic; H. de Waard; H.W. Frijlink; W.L.J. Hinrichs (pp. 763-769).

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominately determines stability at higher glass transition temperatures.► Mechanisms for stabilization of a protein by sugar glasses are investigated. ► The two main mechanisms considered are vitrification and water replacement. ► Both mechanisms are distinguished by varying the glass transition temperature (Tg). ► Vitrification determined stability at Tg's up to 20°C above storage temperature. ► Water replacement determined stability at Tg's well above storage temperature.

Keywords: Alkaline phosphatase; Spray drying; Sugar glass; Vitrification; Water replacement

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant by Barbara Campanini; Barbara Pioselli; Samanta Raboni; Paolo Felici; Immacolata Giordano; Laura D'Alfonso; Maddalena Collini; Giuseppe Chirico; Stefano Bettati (pp. 770-779).

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pK_a values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.► The pK_a of the chromophore increases when H148 of GFPmut2 is mutated to G, K and R. ► H148R and H148K show very low expression yields in the soluble fraction. ► H148G mutation has negligible effects on protein structure and stability in solution. ► An unfolding intermediate is detected on encapsulated H148G GFPmut2. ► All the mutations impact on GFPmut2 dynamics and/or solubility.

Keywords: Abbreviations; GFPmut2; green fluorescent protein mutant 2 (S65A, V68L, S72A GFP); CD; circular dichroism; Mut2G; H148G GFPmut2; Mut2K; H148K GFPmut2; Mut2R; H148R GFPmut2; GdnHCl; guanidinium hydrochlorideGreen fluorescent protein; GFPmut2; Site-directed mutants; Protein dynamics; Unfolding intermediates; Silica gel

Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120 by C. Bhaskar; Palakolanu S. Reddy; K. Sarath Chandra; Sudeep Sabde; Debashis Mitra; Anand K. Kondapi (pp. 780-790).

Early pregnancy associated protein-1 (Epap-1), a 90kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N=8; N=7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region.► Epap-1 and gp120 interaction regions were modeled. ► A 32 mer peptide (P-2) showed significant interaction with gp120. ► P-2 interacts with V3 loop and exhibits anti-HIV-1 activity.

Keywords: Epap-1; HIV-1; Pregnancy; Peptide; gp120-binding

Subcellular fractionation enhances proteome coverage of pancreatic duct cells by Joao A. Paulo; Aleksandr Gaun; Vivek Kadiyala; Ali Ghoulidi; Peter A. Banks; Darwin L. Conwell; Hanno Steen (pp. 791-797).

Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared – via mass spectrometry-based analysis – the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.► SDS-PAGE revealed distinct banding pattern for organelle-enriched fractions. ► Similar numbers of proteins were identified by each method. ► Greater overlap of proteins within replicates than within subcellular fractions. ► Gene ontology revealed expected enrichment of organelle-specific proteins.

Keywords: Organelle enrichment; Chronic pancreatitis; Pancreatic cancer

Analysis of Molecular Recognition Features (MoRFs) in membrane proteins by Ioly Kotta-Loizou; Georgios N. Tsaousis; Stavros J. Hamodrakas (pp. 798-807).

Molecular Recognition Features (MoRFs) are defined as short, intrinsically disordered regions in proteins that undergo disorder-to-order transition upon binding to their partners. As their name suggests, they are implicated in molecular recognition, which serves as the initial step for protein–protein interactions. Membrane proteins constitute approximately 30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. The aim of the current study was to identify and analyze MoRFs in membrane proteins. Two datasets of MoRFs, transmembrane and peripheral membrane protein MoRFs, were constructed from the Protein Data Bank, and sequence, structural and functional analysis was performed. Characterization of our datasets revealed their unique compositional biases and membrane protein MoRFs were categorized depending on their secondary structure after the interaction with their partners. Moreover, the position of transmembrane protein MoRFs in relation with the protein's topology was determined. Further studies were focused on functional analyses of MoRF-containing proteins and MoRFs' partners, associating them with protein binding, regulation and cell signaling, indicating half of them as putative hubs in protein–protein interaction networks. In conclusion, we provide insights into the disorder-based protein–protein interactions involving membrane proteins.Display Omitted► We examined MoRFs in transmembrane and peripheral membrane proteins. ► 60% of MoRFs' residues were in irregular conformation/with missing density. ► Transmembrane protein MoRFs are mostly located on the cytoplasmic segments. ► MoRF-containing proteins are implicated in protein binding and cell signaling. ► MoRF-containing membrane proteins and MoRFs' partners are putative hubs.

Keywords: Abbreviations; MoRFs; Molecular Recognition Features; mpMoRFs; membrane protein MoRFs; non-mpMoRFs; non-membrane protein MoRFs; tmpMoRFs; transmembrane protein MoRFs; pmpMoRFs; peripheral membrane protein MoRFs; lampMoRFs; lipid-anchor membrane protein MoRFs; IDPs; Intrinsically Disordered Proteins; IDRs; Intrinsically Disordered Regions; ELMs; Eukaryotic Linear Motifs; SLiMs; Short Linear MotifsMoRFs; Intrinsic disorder; Protein–protein interactions; Transmembrane proteins; Peripheral membrane proteins

FlgM proteins from different bacteria exhibit different structural characteristics by Wai Kit Ma; Rachel Hendrix; Claire Stewart; Eric V. Campbell; Mitchell Lavarias; Kolyn Morris; Shauna Nichol; Matthew J. Gage (pp. 808-816).

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85°C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.► FlgM orthologs exhibit different predicted degrees of disorder. ► Orthologs of FlgM are in varying extended states. ► FlgM orthologs have different secondary structures. ► Three FlgM orthologs are structurally similar and two orthologs are different.

Keywords: FlgM; Intrinsically disordered protein; Circular dichroism; Molten globule

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BBA - Proteins and Proteomics (v.1834, #4)