Publications by authors named "Emilia Sikorska"

52 Publications

Novel Cell Permeable Polymers of -Substituted L-2,3-Diaminopropionic Acid (DAPEGs) and Cellular Consequences of Their Interactions with Nucleic Acids.

Int J Mol Sci 2021 Mar 4;22(5). Epub 2021 Mar 4.

Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.

The present study aimed to synthesize novel polycationic polymers composed of -substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)-O2Oc-NH, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]-O2Oc-NH and typical for large proteins.
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http://dx.doi.org/10.3390/ijms22052571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961587PMC
March 2021

Biological and Physico-Chemical Characteristics of Arginine-Rich Peptide Gemini Surfactants with Lysine and Cystine Spacers.

Int J Mol Sci 2021 Mar 24;22(7). Epub 2021 Mar 24.

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, 80-416 Gdańsk, Poland.

Ultrashort cationic lipopeptides (USCLs) and gemini cationic surfactants are classes of potent antimicrobials. Our recent study has shown that the branching and shortening of the fatty acids chains with the simultaneous addition of a hydrophobic -terminal amino acid in USCLs result in compounds with enhanced selectivity. Here, this approach was introduced into arginine-rich gemini cationic surfactants. l-cystine diamide and l-lysine amide linkers were used as spacers. Antimicrobial activity against planktonic and biofilm cultures of ESKAPE (, , , , , and spp.) strains and sp. as well as hemolytic and cytotoxic activities were examined. Moreover, antimicrobial activity in the presence of human serum and the ability to form micelles were evaluated. Membrane permeabilization study, serum stability assay, and molecular dynamics were performed. Generally, critical aggregation concentration was linearly correlated with hydrophobicity. Gemini surfactants were more active than the parent USCLs, and they turned out to be selective antimicrobial agents with relatively low hemolytic and cytotoxic activities. Geminis with the l-cystine diamide spacer seem to be less cytotoxic than their l-lysine amide counterparts, but they exhibited lower antibiofilm and antimicrobial activities in serum. In some cases, geminis with branched fatty acid chains and -terminal hydrophobic amino acid resides exhibited enhanced selectivity to pathogens over human cells.
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http://dx.doi.org/10.3390/ijms22073299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036666PMC
March 2021

Investigation of the Effects of Primary Structure Modifications within the RRE Motif on the Conformation of Synthetic Bovine Herpesvirus 1-Encoded UL49.5 Protein Fragments.

Chem Biodivers 2021 Feb 15;18(2):e2000883. Epub 2021 Jan 15.

Faculty of Chemistry, University of Gdańsk, 80-308, Gdańsk, Poland.

Herpesviruses are the most prevalent viruses that infect the human and animal body. They can escape a host immune response in numerous ways. One way is to block the TAP complex so that viral peptides, originating from proteasomal degradation, cannot be transported to the endoplasmic reticulum. As a result, a reduced number of MHC class I molecules appear on the surface of infected cells and, thus, the immune system is not efficiently activated. BoHV-1-encoded UL49.5 protein is one such TAP transporter inhibitor. This protein binds to TAP in such a way that its N-terminal fragment interacts with the loops of the TAP complex, and the C-terminus stimulates proteasomal degradation of TAP. Previous studies have indicated certain amino acid residues, especially the RRE(9-11) motif, within the helical structure of the UL49.5 N-terminal fragment, as being crucial to the protein's activity. In this work, we investigated the effects of modifications within the RRE region on the spatial structure of the UL49.5 N-terminal fragment. The introduced RRE(9-11) variations were designed to abolish or stabilize the structure of the α-helix and, consequently, to increase or decrease protein activity compared to the wild type. The terminal structure of the peptides was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane-mimetic or membrane-model environments. Our structural results show that in the RRE(9-11)AAA and E11G peptides the helical structure has been stabilized, whereas for the RRE(9-11)GGG peptide, as expected, the helix structure has partially unfolded compared to the native structure. These RRE modifications, in the context of the entire UL49.5 proteins, slightly altered their biological activity in human cells.
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http://dx.doi.org/10.1002/cbdv.202000883DOI Listing
February 2021

The Influence of the Mixed DPC:SDS Micelle on the Structure and Oligomerization Process of the Human Cystatin C.

Membranes (Basel) 2020 Dec 24;11(1). Epub 2020 Dec 24.

Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.

Human cystatin C (CC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. Physiologically active CC is a monomer, which dimerization and oligomerization lead to the formation of the inactive, insoluble amyloid form of the protein, strictly associated with cerebral amyloid angiopathy, a severe state causing death among young patients. It is known, that biological membranes may accelerate the oligomerization processes of amyloidogenic proteins. Therefore, in this study, we describe an influence of membrane mimetic environment-mixed dodecylphosphocholine:sodium dodecyl sulfate (DPC:SDS) micelle (molar ratio 5:1)-on the effect of the CC oligomerization. The CC-micelle interactions were analyzed with size exclusion chromatography, circular dichroism, and nuclear magnetic resonance spectroscopy. The experiments were performed on the wild-type (WT) cystatin C, and two CC variants-V57P and V57G. Collected experimental data were supplemented with molecular dynamic simulations, making it possible to highlight the binding interface and select the residues involved in interactions with the micelle. Obtained data shows that the mixed DPC:SDS micelle does not accelerate the oligomerization of protein and even reverses the CC dimerization process.
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http://dx.doi.org/10.3390/membranes11010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824358PMC
December 2020

Double-Headed Cationic Lipopeptides: An Emerging Class of Antimicrobials.

Int J Mol Sci 2020 Nov 25;21(23). Epub 2020 Nov 25.

Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.

Antimicrobial peptides (AMPs) constitute a promising tool in the development of novel therapeutic agents useful in a wide range of bacterial and fungal infections. Among the modifications improving pharmacokinetic and pharmacodynamic characteristics of natural AMPs, an important role is played by lipidation. This study focuses on the newly designed and synthesized lipopeptides containing multiple Lys residues or their shorter homologues with palmitic acid (C) attached to the side chain of a residue located in the center of the peptide sequence. The approach resulted in the development of lipopeptides representing a model of surfactants with two polar headgroups. The aim of this study is to explain how variations in the length of the peptide chain or the hydrocarbon side chain of an amino acid residue modified with C, affect biological functions of lipopeptides, their self-assembling propensity, and their mode of action.
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http://dx.doi.org/10.3390/ijms21238944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728077PMC
November 2020

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity.

Int J Mol Sci 2020 Sep 29;21(19). Epub 2020 Sep 29.

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, 80-416 Gdańsk, Poland.

Ultrashort cationic lipopeptides (USCLs) are considered to be a promising class of antimicrobials with high activity against a broad-spectrum of microorganisms. However, the majority of these compounds are characterized by significant toxicity toward human cells, which hinders their potential application. To overcome those limitations, several approaches have been advanced. One of these is disulfide cyclization that has been shown to improve drug-like characteristics of peptides. In this article the effect of disulfide cyclization of the polar head of -palmitoylated USCLs on in vitro biological activity has been studied. Lipopeptides used in this study consisted of three or four basic amino acids (lysine and arginine) and cystine in a cyclic peptide. In general, disulfide cyclization of the lipopeptides resulted in peptides with reduced cytotoxicity. Disulfide-cyclized USCLs exhibited improved selectivity between sp., Gram-positive strains and normal cells in contrast to their linear counterparts. Interactions between selected USCLs and membranes were studied by molecular dynamics simulations using a coarse-grained force field. Moreover, membrane permeabilization properties and kinetics were examined. Fluorescence and transmission electron microscopy revealed damage to cell membrane and organelles. Concluding, USCLs are strong membrane disruptors and disulfide cyclization of polar head can have a beneficial effect on its in vitro selectivity between sp. and normal human cells.
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http://dx.doi.org/10.3390/ijms21197208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582905PMC
September 2020

Lipidated Analogs of the LL-37-Derived Peptide Fragment KR12-Structural Analysis, Surface-Active Properties and Antimicrobial Activity.

Int J Mol Sci 2020 Jan 30;21(3). Epub 2020 Jan 30.

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, 80-416 Gdańsk, Poland.

An increasing number of multidrug-resistant pathogens is a serious problem of modern medicine and new antibiotics are highly demanded. In this study, different n-alkyl acids (C-C) and aromatic acids (benzoic and -cinnamic) were conjugated to the -terminus of KR12 amide. The effect of this modification on antimicrobial activity (ESKAPE bacteria and biofilm of ) and cytotoxicity (human red blood cells and HaCaT cell line) was examined. The effect of lipophilic modifications on helicity was studied by CD spectroscopy, whereas peptide self-assembly was studied by surface tension measurements and NMR spectroscopy. As shown, conjugation of the KR12-NH peptide with C-C fatty acid chains enhanced the antimicrobial activity with an optimum demonstrated by C-KR12-NH (MIC 1-4 μg/mL against ESKAPE strains; MBEC of 4-16 μg/mL). Correlation between antimicrobial activity and self-assembly behavior of C-KR12-NH and C-KR12-NH has shown that the former self-assembled into larger aggregated structures, which reduced its antimicrobial activity. In conclusion, -terminal modification can enhance antimicrobial activity of KR12-NH; however, at the same time, the cytotoxicity increases. It seems that the selectivity against pathogens over human cells can be achieved through conjugation of peptide -terminus with appropriate n-alkyl fatty and aromatic acids.
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http://dx.doi.org/10.3390/ijms21030887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036753PMC
January 2020

The motif EEL in the cytosolic tail of the secretory human proprotein convertase PC7 regulates its trafficking and cleavage activity.

J Biol Chem 2020 02 8;295(7):2068-2083. Epub 2020 Jan 8.

Laboratory of Biochemical Neuroendocrinology, Clinical Research of Montreal, affiliated with Université de Montréal, Montreal, Quebec H2W 1R7, Canada. Electronic address:

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the -Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EEL, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EEL motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.
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http://dx.doi.org/10.1074/jbc.RA119.011775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029121PMC
February 2020

Effect of self-assembly on antimicrobial activity of double-chain short cationic lipopeptides.

Bioorg Med Chem 2019 12 17;27(23):115129. Epub 2019 Oct 17.

Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. Electronic address:

Short cationic antimicrobial lipopeptides with surfactant-like structure are promising antibiotic candidates that preferentially target microbial membranes. Therefore, we focused our study on double-chain lipopeptides, (C)Dab-KKK-NH and (C)Dap-KKK-NH, where Dab and Dap are 2,4-diaminobutyric and 2,3-diaminopropionic acids, respectively. We tried to answer a question how the self-assembly behaviour affects biological activities of the tested compounds. The subject compounds were synthesized by solid-phase method and screened for their antimicrobial and haemolytic activities. Cytotoxicity tests on human keratinocytes were carried out for the most promising lipopeptides. Self-assembly properties were evaluated by both experimental and theoretical methods. Interactions with membrane models were examined using the ITC and FTIR techniques. All the lipopeptides studied showed the tendency to self-assembly in solution, and this behaviour was affected by the length of the hydrocarbon chains. Acyl chain elongation supported the formation of the bilayer structure and deprived the lipopeptides of antimicrobial activity. A multi-step mechanism of interaction with a negatively charged membrane was observed for the short-chain lipopeptides, indicating other processes accompanying the binding process. Short-chain lipopeptides were able to penetrate into the liposome's interior and/or cause the rupture of the liposome, this being compatible with their high antimicrobial activity.
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http://dx.doi.org/10.1016/j.bmc.2019.115129DOI Listing
December 2019

Structural Analysis and Dynamic Processes of the Transmembrane Segment Inside Different Micellar Environments-Implications for the TM4 Fragment of the Bilitranslocase Protein.

Int J Mol Sci 2019 Aug 26;20(17). Epub 2019 Aug 26.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

The transmembrane (TM) proteins are gateways for molecular transport across the cell membrane that are often selected as potential targets for drug design. The bilitranslocase (BTL) protein facilitates the uptake of various anions, such as bilirubin, from the blood into the liver cells. As previously established, there are four hydrophobic transmembrane segments (TM1-TM4), which constitute the structure of the transmembrane channel of the BTL protein. In our previous studies, the 3D high-resolution structure of the TM2 and TM3 transmembrane fragments of the BTL in sodium dodecyl sulfate (SDS) micellar media were solved using Nuclear Magnetic Resonance (NMR) spectroscopy and molecular dynamics simulations (MD). The high-resolution 3D structure of the fourth transmembrane region (TM4) of the BTL was evaluated using NMR spectroscopy in two different micellar media, anionic SDS and zwitterionic DPC (dodecylphosphocholine). The presented experimental data revealed the existence of an α -helical conformation in the central part of the TM4 in both micellar media. In the case of SDS surfactant, the α -helical conformation is observed for the Pro258-Asn269 region. The use of the zwitterionic DPC micelle leads to the formation of an amphipathic α -helix, which is characterized by the extension of the central α -helix in the TM4 fragment to Phe257-Thr271. The complex character of the dynamic processes in the TM4 peptide within both surfactants was analyzed based on the relaxation data acquired on 15 N and 31 P isotopes. Contrary to previously published and present observations in the SDS micelle, the zwitterionic DPC environment leads to intensive low-frequency molecular dynamic processes in the TM4 fragment.
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http://dx.doi.org/10.3390/ijms20174172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747479PMC
August 2019

Synthesis and SAR Studies of Antibacterial Peptidyl Derivatives Based upon the Binding Site of Human Cystatin C.

Protein Pept Lett 2019 Jul;26(6):423-434

Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland.

Background: Antibacterial peptidyl derivative - Cystapep 1, was previously found to be active both against antibiotic-resistant staphylococci and streptococci as well as antibioticsusceptible strains of these species. Therefore, it is a promising lead compound to search for new antimicrobial peptidomimetics.

Objectives: We focused on identifying structural elements that are responsible for the biological activity of Cystapep 1 and its five analogues. We tried to find an answer to the question about the mechanism of action of the tested compounds. Therefore, we have investigated in details the possibility of interacting these compounds with biological membrane mimetics.

Methods: The subject compounds were synthesized in solution, purified and characterized by HPLC and mass spectrometry. Then, the staphylococci susceptibility tests were performed and their cytotoxicity was established. The results of Cystapep 1 and its analogues interactions with model target were examined using the DSC and ITC techniques. At the end the spatial structures of the tested peptidomimetics using NMR technique were obtained.

Results: Antimicrobial and cytotoxicity tests show that Cystapep 1 and its peptidomimetic V are good drug candidates. DSC and ITC studies indicate that disruption of membrane is not the only possible mechanism of action of Cystapep 1-like compounds. For Cystapep 1 itself, a multi-step mechanism of interaction with a negatively charged membrane is observed, which indicates other processes occurring alongside the binding process. The conformational analysis indicated the presence of a hydrophobic cluster, composed of certain side chains, only in the structures of active peptidomimetics. This can facilitate the anchoring of the peptidyl derivatives to the bacterial membrane.

Conclusion: An increase in hydrophobicity of the peptidomimetics improved the antimicrobial activity against S. aureus, however there is no simple correlation between the biological activity and the strength of interactions of the peptidyl with bacterial membrane.
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http://dx.doi.org/10.2174/0929866526666190311162716DOI Listing
July 2019

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics.

Biochim Biophys Acta Biomembr 2019 05 14;1861(5):926-938. Epub 2019 Feb 14.

Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland. Electronic address:

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far. Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembrane-intracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer. Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections.
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http://dx.doi.org/10.1016/j.bbamem.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089609PMC
May 2019

Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2.

Probiotics Antimicrob Proteins 2019 09;11(3):1042-1054

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416, Gdańsk, Poland.

Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chromatography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.
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http://dx.doi.org/10.1007/s12602-018-9501-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695355PMC
September 2019

Short arginine-rich lipopeptides: From self-assembly to antimicrobial activity.

Biochim Biophys Acta Biomembr 2018 11 7;1860(11):2242-2251. Epub 2018 Sep 7.

Faculty of Pharmacy, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland.

In this paper, we examine antimicrobial and cytotoxic activities, self-assembly and interactions with anionic and zwitterionic membranes of short arginine-rich lipopeptides: C-RRRR-NH, C-RRRR-NH, C-RRRR-NH, and C-PRRR-NH. They show a tendency to self-assembly into micelles, but it is not required for antimicrobial activity. The membrane binding of the lipopeptides can be accompanied by other factors such as: peptide aggregation, pore formation or micellization of phospholipid bilayer. The shortening of the acyl chain results in compounds with a lower haemolytic activity and a slightly improved antimicrobial activity against Gram-positive bacteria, what indicates enhanced cell specificity. Results of coarse-grained molecular dynamics simulations indicate different organization of membrane lipids upon binding of arginine-based lipopeptides and the previously studied lysine-based ones.
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http://dx.doi.org/10.1016/j.bbamem.2018.09.004DOI Listing
November 2018

[The role of proprotein convertases in cancer diseases with particular focus on PACE4].

Postepy Biochem 2017;63(3):179-184

Laboratory of Peptide Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Gdańsk, 63 Wita Stwosza St., 80-308 Gdansk, Poland.

Cancer is one of the most common cause of death nowadays. Thorough knowledge of the mechanisms of tumorigenesis and invasiveness of tumor cells is crucial for the development of molecular targeted therapies, which are believed to be future treatment of this type of diseases. Proteolytic enzymes are one of the factors involved in the development of cancer cells, very often used as markers of tumor progression. In this paper we describe the role of enzymes termed proprotein convertases (PCs) in pathogenesis and progress of cancer diseases. Furthermore, we indicate potential directions for the development of therapeutic strategies designed based on PCs inhibitors.
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May 2019

Corrigendum to "Rapid PD-L1 detection in tumors with PET using a highly specific peptide" [Biochemical and Biophysical Research Communications 483/1 (2017) 258-263].

Biochem Biophys Res Commun 2017 09 8;491(4):1125. Epub 2017 Aug 8.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, USA; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. Electronic address:

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http://dx.doi.org/10.1016/j.bbrc.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600467PMC
September 2017

Retro analog concept: comparative study on physico-chemical and biological properties of selected antimicrobial peptides.

Amino Acids 2017 10 29;49(10):1755-1771. Epub 2017 Jul 29.

Faculty of Pharmacy, Medical University of Gdańsk, Gdańsk, Poland.

Increasing drug resistance of common pathogens urgently needs discovery of new effective molecules. Antimicrobial peptides are believed to be one of the possible solutions of this problem. One of the approaches for improvement of biological properties is reversion of the sequence (retro analog concept). This research is based on investigation of antimicrobial activity against Gram-positive, Gram-negative bacteria, and fungi, hemolysis of erythrocytes, interpretation of the circular dichroism spectra, measurement of counter-ion content, and assessment of the peptide hydrophobicity and self-assembly using reversed-phase chromatography. The experiments were conducted using the following peptides: aurein 1.2, CAMEL, citropin 1.1, omiganan, pexiganan, temporin A, and their retro analogs. Among the compounds studied, only retro omiganan showed an enhanced antimicrobial and a slightly increased hemolytic activity as compared to parent molecule. Moreover, retro pexiganan exhibited high activity towards Klebsiella pneumoniae, whereas pexiganan was in general more or equally active against the rest of tested microorganisms. Furthermore, the determined activity was closely related to the peptide hydrophobicity. In general, the reduced hemolytic activity correlates with lower antimicrobial activity. The tendency to self-association and helicity fraction in SDS seems to be correlated. The normalized RP-HPLC-temperature profiles of citropin 1.1 and aurein 1.2, revealed an enhanced tendency to self-association than that of their retro analogs.
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http://dx.doi.org/10.1007/s00726-017-2473-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602100PMC
October 2017

[Proprotein convertases - family of serine proteases with a broad spectrum of physiological functions].

Postepy Biochem 2016;62(4):472-481

Laboratory of Peptide Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Gdańsk, 63 Wita Stwosza St., 80-308 Gdansk, Poland.

A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.
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December 2017

Rapid PD-L1 detection in tumors with PET using a highly specific peptide.

Biochem Biophys Res Commun 2017 01 24;483(1):258-263. Epub 2016 Dec 24.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, USA; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA. Electronic address:

Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration.
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http://dx.doi.org/10.1016/j.bbrc.2016.12.156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253331PMC
January 2017

Antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak.

J Pept Sci 2016 Nov 9;22(11-12):723-730. Epub 2016 Nov 9.

Medical University of Gdansk, Faculty of Pharmacy, Gdansk, Poland.

This is the study on the effect of opiorphin, sialorphin and their analogs on antitumor activity. We demonstrated that conjugation of opiorphin and sialorphin with a proapoptotic, antimicrobial peptide klak (klaklakklaklak) led to compounds (opio-klak and sialo-klak) that were cytotoxic against cancer cells (LN18, PC3, A549, HCT116 and B10-F16) in the MTT test. The conjugated analogs were designed to increase the effectiveness of the peptide. The opio-klak derivative was the most effective in the in vitro assays and led to a decrease in viability of cancer cells over time as compared with that of untreated controls. In contrast, treatment with either the untargeted klak peptide or opiorphin as a negative control led to a negligible loss in viability. Antitumor effect of the opio-klak was also observed in vivo in murine melanoma tumor-bearing mice. Cessation of peptide administration resulted in tumor regrowth. Our results are seemingly valuable for the development of opiorphin analogs with potential clinical applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.2936DOI Listing
November 2016

Potent antidiuretic agonists, deamino-vasopressin and desmopressin, and their inverso analogs: NMR structure and interactions with micellar and liposomic models of cell membrane.

Biopolymers 2016 May;106(3):245-59

Faculty of Chemistry, University of Gdańsk, Gdańsk, 80-308, Poland.

Deamination of vasopressin (AVP) enhances its antidiuretic activity. Moreover, introduction of D-Arg8 instead of its L enantiomer in deamino-vasopressin (dAVP) results in an extremely potent and selective antidiuretic agonist - desmopressin (dDAVP). In this study we describe the synthesis, pharmacological properties and structures of these two potent antidiuretic agonists, and their inverso analogs. The structures of the peptides are studied in micellar and liposomic models of cell membrane using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy supported by molecular dynamics simulations. Our conformational studies have shown that desmopressin in a membrane mimicking environment adopts one of the characteristic for vasopressin-like peptides β-turn - in position 3,4. Furthermore, dDAVP shows the tendency to create a β-turn in the Cys6-Gly9 C-tail, considered to be important for the antidiuretic activity, and also some tendency to adopt a 5,6 β-turn. In desmopressin, in contrast to the native vasopressin, deamino-vasopressin and [D-Arg8]-vasopressin (DAVP), the Arg8 side chain, crucial for the pressor and antidiuretic activities, is very well exposed for interaction with the receptor, whereas Gly9, crucial for the pressor and uterotonic activities, is situated together with the C-terminal amide group very close to the tocin ring. The arrangements of the Gln4 and Asn5 side chains, being crucial for OT activity, also differ in desmopressin as compared to those of AVP, dAVP and DAVP. These differences in arrangement of the important for activities side chains are likely to explain extremely potent and selective antidiuretic activities of desmopressin. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 245-259, 2016.
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http://dx.doi.org/10.1002/bip.22825DOI Listing
May 2016

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

PLoS One 2015 17;10(11):e0143038. Epub 2015 Nov 17.

Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648528PMC
June 2016

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data.

PLoS One 2015 20;10(8):e0135455. Epub 2015 Aug 20.

National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia.

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546402PMC
May 2016

Arginine-, D-arginine-vasopressin, and their inverso analogues in micellar and liposomic models of cell membrane: CD, NMR, and molecular dynamics studies.

Eur Biophys J 2015 Dec 20;44(8):727-43. Epub 2015 Aug 20.

Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308, Gdańsk, Poland.

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [D-Arg(8)]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides β-turns: in the 2-5 and 3-6 fragments. The inverso-analogues also adopt β-turns in the 3-6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr(2), Phe(3), and Arg(8), important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr(2) and Phe(3) in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide-membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic-zwitterionic liposomes as well as the anionic-zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.
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http://dx.doi.org/10.1007/s00249-015-1071-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628624PMC
December 2015

Influence of Dimerization of Lipopeptide Laur-Orn-Orn-Cys-NH and an -terminal Peptide of Human Lactoferricin on Biological Activity.

Int J Pept Res Ther 2015;21:39-46. Epub 2014 Aug 21.

Faculty of Pharmacy, Medical University of Gdańsk, Al. Gen. Hallera 107, 80-416 Gdańsk, Poland.

Lactoferrin (LF) is a naturally occurring antimicrobial peptide that is cleaved by pepsin to lactoferricin (LFcin). LFcin has an enhanced antimicrobial activity as compared to that of LF. Recently several hetero- and homodimeric antimicrobial peptides stabilized by a single disulfide bond linking linear polypeptide chains have been discovered. We have demonstrated that the S-S bond heterodimerization of lipopeptide Laur-Orn-Orn-Cys-NH (peptide III) and the synthetic -terminal peptide of human lactoferricin (peptide I) yields a dimer (peptide V), which is almost as microbiologically active as the more active monomer and at the same time it is much less toxic. Furthermore, it has been found that the S-S bond homodimerization of both peptide I and peptide III did not affect antimicrobial and haemolytic activity of the compounds. The homo- and heterodimerization of peptides I and III resulted in either reduction or loss of antifungal activity. This work suggests that heterodimerization of antimicrobial lipopeptides via intermolecular disulfide bond might be a powerful modification deserving consideration in the design of antimicrobial peptides.
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http://dx.doi.org/10.1007/s10989-014-9423-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305368PMC
August 2014

Structural characterization and in vivo evaluation of β-Hairpin peptidomimetics as specific CXCR4 imaging agents.

Mol Pharm 2015 Mar 3;12(3):941-53. Epub 2015 Feb 3.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University , Baltimore, Maryland 21287, United States.

The CXCR4 chemokine receptor is integral to several biological functions and plays a pivotal role in the pathophysiology of many diseases. As such, CXCR4 is an enticing target for the development of imaging and therapeutic agents. Here we report the evaluation of the POL3026 peptidomimetic template for the development of imaging agents that target CXCR4. Structural and conformational analyses of POL3026 and two of its conjugates, DOTA (POL-D) and PEG12-DOTA (POL-PD), by circular dichroism, two-dimensional NMR spectroscopy and molecular dynamics calculations are reported. In silico observations were experimentally verified with in vitro affinity assays and rationalized using crystal structure-based molecular modeling studies. [(111)In]-labeled DOTA conjugates were assessed in vivo for target specificity in CXCR4 expressing subcutaneous U87 tumors (U87-stb-CXCR4) through single photon emission computed tomography (SPECT/CT) imaging and biodistribution studies. In silico and in vitro studies show that POL3026 and its conjugates demonstrate similar interactions with different micelles that mimic cellular membrane and that the ε-NH2 of lysine(7) is critical to maintain high affinity to CXCR4. Modification of this group with DOTA or PEG12-DOTA led to the decrease of IC50 value from 0.087 nM for POL3026 to 0.47 nM and 1.42 nM for POL-D and POL-PD, respectively. In spite of the decreased affinity toward CXCR4, [(111)In]POL-D and [(111)In]POL-PD demonstrated high and significant uptake in U87-stb-CXCR4 tumors compared to the control U87 tumors at 90 min and 24 h post injection. Uptake in U87-stb-CXCR4 tumors could be blocked by unlabeled POL3026, indicating specificity of the agents in vivo. These results suggest POL3026 as a promising template to develop new imaging agents that target CXCR4.
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http://dx.doi.org/10.1021/mp500799qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612581PMC
March 2015

Effect of head-to-tail cyclization on conformation of histatin-5.

J Pept Sci 2014 Dec 31;20(12):952-7. Epub 2014 Oct 31.

Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308, Gdansk, Poland.

Histatin-5 (Hst-5, DSHAKRHHGYKRKFHEKHHSHRGY) is a member of a histidine-rich peptide family secreted by major salivary glands, exhibiting high fungicidal activity against Candida albicans. In the present work, we demonstrate the 3D structure of the head-to-tail cyclic variant of Hst-5 in TFE solution determined using NMR spectroscopy and molecular dynamics simulations. The cyclic histatin-5 reveals a helix-loop-helix motif with α-helices at positions Ala(4)-His(7) and Lys(11)-Ser(20). Both helical segments are arranged relative to each other at an angle of ca. 142°. The head-to-tail cyclization increases amphipathicity of the peptide, this, however, does not affect its antimicrobial potency.
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http://dx.doi.org/10.1002/psc.2707DOI Listing
December 2014

Self-assembly and interactions of short antimicrobial cationic lipopeptides with membrane lipids: ITC, FTIR and molecular dynamics studies.

Biochim Biophys Acta 2014 Oct 28;1838(10):2625-34. Epub 2014 Jun 28.

Faculty of Pharmacy with Subfaculty of Laboratory Medicine, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland.

In this work, the self-organization and the behavior of the surfactant-like peptides in the presence of biological membrane models were studied. The studies were focused on synthetic palmitic acid-containing lipopeptides, C16-KK-NH2 (I), C16-KGK-NH2 (II) and C16-KKKK-NH2 (III). The self-assembly was explored by molecular dynamics simulations using a coarse-grained force field. The critical micellar concentration was estimated by the surface tension measurements. The thermodynamics of the peptides binding to the anionic and zwitterionic lipids were established using isothermal titration calorimetry (ITC). The influence of the peptides on the lipid acyl chain ordering was determined using FTIR spectroscopy. The compounds studied show surface-active properties with a distinct CMC over the millimolar range. An increase in the steric and electrostatic repulsion between polar head groups shifts the CMC toward higher values and reduces the aggregation number. An analysis of the peptide-membrane binding revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions enabling the lipopeptides to interact with the lipid bilayer. In the case of C16-KKKK-NH2 (III), compensation of the electrostatic and hydrophobic interactions upon binding to the anionic membrane has been suggested and consequently no overall binding effects were noticed in ITC thermograms and FTIR spectra.
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http://dx.doi.org/10.1016/j.bbamem.2014.06.016DOI Listing
October 2014

Conformational studies of neurohypophyseal hormones analogues with glycoconjugates by NMR spectroscopy.

J Pept Sci 2014 Jun 18;20(6):406-14. Epub 2014 Mar 18.

Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952, Gdańsk, Poland.

Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α-d-mannose through the oxygen β atom, whereas peptide II is a lysine-vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β-turn characteristic of OT-like peptides, and vasopressin-glycosylated analogue (II) exhibits β-turns typical of vasopressin-like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT-like and vasopressin-like drugs.
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http://dx.doi.org/10.1002/psc.2628DOI Listing
June 2014

Structural elucidation of transmembrane transporter protein bilitranslocase: conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.

Biochim Biophys Acta 2013 Nov 15;1828(11):2609-19. Epub 2013 Jun 15.

National Institute of Chemistry, Ljubljana, Slovenia. Electronic address:

Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds.
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http://dx.doi.org/10.1016/j.bbamem.2013.06.006DOI Listing
November 2013