Structure Effects of Double D-Amino Acid Replacements: A Nuclear Magnetic Resonance and Circular Dichroism Study Using Amphipathic Model Helixes

Rothemund, Sven; Beyermann, Michael; Krause, Eberhard; Krause, Gerd; Bienert, Michael; Rs, Hodges; Bd, Sykes; Sönnichsen, Frank D.

doi:10.1021/bi00040a005

Cited by 69 publications

(62 citation statements)

References 41 publications

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“…The lipid SM was not used because it has a strong signal in the amide I region, but the results in the functional assays were similar when using liposomes with or without SM. Our assignment of the different secondary structures was the same as that described previously (15)(16)(17)(18)(19)(20). The data revealed that the peptide adopts predominantly distorted/dynamic helical structures in both types of lipids ( Fig.…”

Section: Anticancer Activity Insupporting

confidence: 73%

A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

Papo

Shahar

Eisenbach

et al. 2003

Journal of Biological Chemistry

145

113

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The high toxicity of most chemotherapeutic drugs and their inactivation by multidrug resistance phenotypes motivated extensive search for drugs with new modes of action. We designed a short cationic diastereomeric peptide composed of D-and L-leucines, lysines, and arginines that has selective toxicity toward cancer cells and significantly inhibits lung metastasis formation in mice (86%) with no detectable side effects. Its ability to depolarize the transmembrane potential of cancer cells at the same rate (within minutes) and concentration (3 M), at which it shows biological activity, suggests a killing mechanism that involves plasma membrane perturbation. Confocal microscopy experiments verified that the cells died as a result of acute injury, swelling, and bursting, suggesting necrosis. Biosensor binding experiments and attenuated total reflectance-Fourier transform infrared spectroscopy using model membranes have substantiated its high selectivity toward cancer cells. Although this is an initial study that looked at tumor formation rather than the ability of the peptides to reduce established tumors, the simple sequence of the peptide, its high solubility, substantial resistance to degradation, and inactivation by serum components might make it a good candidate for future anticancer treatment.Current anticancer chemotherapies that are based on alkylating agents, antimetabolites, and natural products respond incompletely; this is probably related to the development of drug resistance. Most chemotherapeutic agents also affect normal cells and consequently cause severe side effects (1). Thus, there is an urgent need to develop new classes of anticancer drugs with new modes of action that selectively target the cancer cells. A promising group under investigation includes cationic antimicrobial peptides, which are known to play an important role in the innate immunity of a diverse range of organisms, including insects, amphibians, and mammals (2). Most of these peptides have an amphipathic structure, and they preferentially bind and insert into negatively charged cell membranes. Consequently, destabilization of the membrane disturbs the electrolyte balance and induces the intracellular contents to leak, leading to cell death. In contrast to normal eukaryotic cells, which generally have low membrane potentials and whose outer leaflet almost exclusively consists of zwitterionic phospholipids, the prokaryotic and cancer cell membranes maintain large transmembrane potentials and have a higher content of anionic phospholipids on their outer leaflet. Many antimicrobial peptides therefore preferentially disrupt prokaryotic and cancer cell membranes rather than eukaryotic membranes (3).Several in vitro studies were conducted with native all L-amino acid antimicrobial peptides with defined ␣-helical or ␤-sheet secondary structures (3). However, the use of native antimicrobial peptides in vivo is mainly limited due to the loss of their function in serum, partially because of enzymatic degradation and binding to serum compone...

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Section: Anticancer Activity Insupporting

confidence: 73%

A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

Papo

Shahar

Eisenbach

et al. 2003

Journal of Biological Chemistry

145

113

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“…Prior to preparing the samples, the trifluoroacetate (CF3COO Ϫ ) counterions, which strongly associate with the peptide, were replaced with chloride ions by washing in 0.1 M HCl and lyophilization. This eliminated the strong C ϭ O stretching absorption band near 1673 cm Ϫ1 (34). Lipid/peptide mixtures were prepared by dissolving the peptide in trifluoroethanol and the lipid in a 1:2 MeOH: CH 2 Cl 2 mixture and drying them under vacuum for 15 min.…”

Section: Construction Of the Toxrmentioning

confidence: 99%

The Identification of a Minimal Dimerization Motif QXXS That Enables Homo- and Hetero-association of Transmembrane Helices in Vivo

Sal‐Man

Gerber

Shai

2005

Journal of Biological Chemistry

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Assembly of transmembrane (TM) domains is a critical step in the function of membrane proteins, and therefore, determining the amino acid motifs that mediate this process is important. Studies along this line have shown that the GXXXG motif is involved in TM assembly. In this study we characterized the minimal dimerization motif in the bacterial Tar-1 homodimer TM domain, which does not contain a GXXXG sequence. We found that a short polar motif QXXS is sufficient to induce stable TM-TM interactions. Statistical analysis revealed that this motif appears to be significantly overrepresented in a bacterial TM data base compared with its theoretical expectancy, suggesting a general role for this motif in TM assembly. A truncated short TM peptide (9 residues) that contains the QXXS motif interacted slightly with the wild-type Tar-1. However, the same short TM peptide regained wild-type-like activity when conjugated to an octanoyl aliphatic moiety. Biophysical studies indicated that this modification compensated for the missing hydrophobicity, stabilized ␣-helical structure, and enabled insertion of the peptide into the membrane core. These findings serve as direct evidence that even a short peptide containing a minimal recognition motif is sufficient to inhibit the proper assembly of TM domains. Interestingly, electron microscopy revealed that above the critical micellar concentration, the TM lipopeptide forms a network of nanofibers, which can serve for the slow release of the active lipopeptide.The function of many proteins can be regulated by alteration of their oligomerization state. For example, information that the cells receive from the outside is thought to be communicated to cells via changes in the oligomerization of membrane receptor proteins. This information is crucial for important cellular processes such as homeostasis and signal transduction (1-6). Receptor oligomerization is mainly mediated by the extracellular or intracellular domains. However, considerable data have been accumulated concerning the causal involvement of the transmembrane (TM) 1 domains in this process as well (7-11). In contrast to the soluble regions of membrane proteins, our knowledge of the factors that control proteinprotein interaction and recognition of the membrane-embedded domains is still limited. The only TM domain dimerization motif identified to date is the GXXXG sequence (11-14). However, there are many examples of TM domains that assemble even though they do not contain the GXXXG motif. One such example is the N-terminal TM domain of the Escherichia coli aspartate receptor (Tar). This receptor is one of the main chemotaxis receptors found in bacteria, and it forms a homodimer complex in which each subunit is composed of two TM helices (Tar-1 and Tar-2) separated by a substantial periplasmic domain. Biochemical studies demonstrated that the N-terminal TM domain of the receptor (Tar-1) is able to dimerize and therefore contributes to the dimerization of the protein (15-17), although dimerization is mainly driven by its p...

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“…23,24 We included analogs showing conformational disturbances via double D amino acid substitutions since replacement of two adjacent amino acids by their D isomers has been shown to destabilize both a-helices and b-sheet structures without changing other properties of the peptide such as hydrophobicity, side chain functionality or charge distribution. 24,25 The data show that structure-breaking D amino acid substitutions lead to improved MALDI signal intensities indicating that the propensity of peptides to adopt ahelical and particularly b-sheet structures may result in diminished MALDI sensitivity. …”

mentioning

confidence: 99%

The ease of peptide detection by matrix-assisted laser desorption/ionization mass spectrometry: the effect of secondary structure on signal intensity

Wenschuh

Halada

Lamer

et al. 1998

Rapid Commun. Mass Spectrom.

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Several structurally well-characterized model peptides were used to examine the relationship between peptide structure and signal intensity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that peptides displaying stable alpha-helical and beta-sheet structures show lower signal intensities than the corresponding analogs having disturbed secondary structures caused by substitution of two adjacent amino acids by their D isomers. Since such substitutions do not affect properties other than the secondary structure propensity, the differences observed are ascribed to this phenomenon or some related effect such as association. The results indicate that the formation of stable secondary structures in peptides may be a possible source of incomplete peptide mass fingerprints resulting from protein digestion and for difficulties in the quantitative evaluation of peptide mixtures via MALDI-MS.

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Structure Effects of Double D-Amino Acid Replacements: A Nuclear Magnetic Resonance and Circular Dichroism Study Using Amphipathic Model Helixes

Cited by 69 publications

References 41 publications

A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

The Identification of a Minimal Dimerization Motif QXXS That Enables Homo- and Hetero-association of Transmembrane Helices in Vivo

The ease of peptide detection by matrix-assisted laser desorption/ionization mass spectrometry: the effect of secondary structure on signal intensity

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