Solvent effects on the secondary structure of the membrane-active zervamicin determined by PELDOR spectroscopy

Zervamicin is a voltage-gated ion-channel-forming peptide. Channels are generally considered to be formed by first insertion of amphipathic molecules into the phospholipid bilayer, followed by self-assembly of a variable number of transmembrane helices. We have studied the length of the peptide structure to address the question whether this peptide is long enough to span the phospholipid bilayer. The pulsed electron-electron double resonance (PELDOR) spectroscopic technique was used to determine the length of the helical molecule in membrane-mimicking solvents. This was achieved from the distance-related dipole-dipole interaction between spin labels, which were located at both ends of the linear peptide chain. The data were obtained by using samples of frozen glassy solutions of MeOH, MeOH/toluene, and MeOH/CHCl(3). Contributions of inter- and intramolecular interactions of spin labels were separated to analyze the intramolecular interaction and the distance distribution function between the labels. It is shown that the main maximum of the distribution functions is located at a distance of ca. 3.3 nm, and this distance appears to be only slightly dependent on the solvent composition. The distribution function was observed to narrow after addition of either CHCl(3) or toluene to MeOH. This effect is rationalized in terms of a decreased mobility of the terminal amino acid residues. By molecular-dynamics simulations, it was shown that the conformation, corresponding with the predominant distance found by PELDOR, agrees well with the mixed alpha/3(10)-helical that was previously determined by NMR. However, in the case toluene was added to the MeOH solution to further increase the hydrophobicity of the environment of the membrane-active peptide, the distribution function gives rise to a minor fraction (7-8%) with a distance of 4.2 nm. This distance corresponds most likely to the more extended 2(7)-helix structure.     

Total syntheses in solution of TOAC-labelled alamethicin F50/5 analogues

Total syntheses in solution of a set of four selected analogues of the 19-mer component F50/5 of alamethicin, the most extensively studied among the channel-former peptaibol antibiotics, are planned and reported. All analogues bear three Glu(OMe) residues, replacing the Gln residues at positions 7, 18, and 19 of the naturally occurring compound. Three analogues are mono-labelled with the free-radical-containing amino acid residue TOAC at the strategic positions 1, 8, or 16. The fourth analogue is bis-labelled with the same EPR-active residue at both positions 1 and 16. In the native sequence, all of the positions where TOAC replacements have been introduced are characterized by residues of Aib, the prototype of the class of helicogenic C(alpha)-tetrasubstituted alpha-amino acids. All of the TOAC analogues synthesized exhibit significant membrane-modifying properties.     

应用ESR检测探讨急性热暴露大鼠肝脏氧化还原状态的动态变化

目的:通过电子顺磁自旋共振技术(ESR)动态观察大鼠在过热条件下肝脏的氧化还原状态.方法:将52只雄性Wistar大鼠随机分成4组:①加温组:麻醉后进行整体加温到直肠温达(43.0±0.5)℃,持续15 min;②对照组:只进行麻醉处理;③,MPG预处理组:用抗氧化剂MPG预处理后,再进行与上述①同样条件的加温处理;④非MPG预处理组:在③中用生理盐水代替MPG.经过以上处理后在不同时间点取肝脏制备组织匀浆,测定ESR波谱.结果:与对照组比较,加温组热暴露处理后记录的ESR波谱振幅-时间直线斜率增大,2 h达最大值.以后逐渐恢复,24 h接近对照组水平.经抗氧化剂预处理上述反应减弱.结论:过热能诱导肝脏产生活性氧,增强其氧化还原反应.     

乙酸乙酯抽提法在ESR检测一氧化氮自由基中的应用

改进了用有机溶剂抽提检测一氧化氮(nitric oxide,NO)自由基的方法,并利用有机溶剂抽提法检测了小鼠心肌中NO的含量.有机溶剂可以把二乙基二硫代氨基甲酸钠(diethyldithiocarbamate, DETC)捕集NO的产物(DETC)₂-Fe²⁺-NO由水相中萃取并富集到酯相中,然后利用电子顺磁共振波谱仪(ESR)在常温下检测大体积样品中的NO.比较了几种不同的有机溶剂：正丁醇、乙酸丁酯、乙酸乙酯、三乙酸甘油酯、乙酸异戊酯等的萃取能力,发现乙酸乙酯是一种理想的提取溶剂.乙酸乙酯提取法可以使NO的量与ESR信号强度在20 μmol/L内有良好的线性,使ESR的检测灵敏度提高到200 nmol/L以下;(DETC)₂-Fe²⁺-NO对光比较敏感,见光易于分解;复合物在乙酸乙酯中避光保存于4℃可稳定十几天而无显著的变化.     

The Impact of Model Peptides on Structural and Dynamic Properties of Egg Yolk Lecithin Liposomes – Experimental and DFT Studies

Electron spin resonance (ESR), ¹H‐NMR, voltage and resistance experiments were performed to explore structural and dynamic changes of Egg Yolk Lecithin (EYL) bilayer upon addition of model peptides. Two of them are phenylalanine (Phe) derivatives, Ac‐Phe‐NHMe ( 1 ) and Ac‐Phe‐NMe₂ ( 2 ), and the third one, Ac‐(Z)‐ΔPhe‐NMe₂ ( 3 ), is a derivative of (Z)‐α,β‐dehydrophenylalanine. The ESR results revealed that all compounds reduced the fluidity of liposome's membrane, and the highest activity was observed for compound 2 with N‐methylated C‐terminal amide bond (Ac‐Phe‐NMe₂). This compound, being the most hydrophobic, penetrates easily through biological membranes. This was also observed in voltage and resistance studies. ¹H‐NMR studies provided a sound evidence on H‐bond interactions between the studied diamides and lecithin polar head. The most significant changes in H‐atom chemical shifts and spin‐lattice relaxation times T₁ were observed for compound 1 . Our experimental studies were supported by theoretical calculations. Complexes EYL? Ac‐Phe‐NMe₂ and EYL? Ac‐(Z)‐ΔPhe‐NMe₂, stabilized by NH???O or/and CH???O H‐bonds were created and optimized at M06‐2X/6‐31G(d) level of theory in vacuo and in H₂O environment. According to our molecular‐modeling studies, the most probable lecithin site of H‐bond interaction with studied diamides is the negatively charged O‐atom in phosphate group which acts as H‐atom acceptor. Moreover, the highest binding energy to hydrocarbon chains were observed in the case of Ac‐Phe‐NMe₂ ( 2 ).     

The production and characterisation of trichotoxin peptaibols, by Trichoderma asperellum

Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.     

Membrane permeabilization of a mammalian neuroendocrine cell type (PC12) by the channel-forming peptides zervamicin, alamethicin, and gramicidin

Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram-positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1 muM GRAM addition to PC12 cells kept at a membrane potential V(m)=0 mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. -24 mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5-10 microM concentrations while V(m) is kept at -60 mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1-2 min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10-20 min at V(m)=0 mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K(+)-release measurements from a PC12 suspension with a K(+)-selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis-positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy.

An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at $\text{5.1 · 10}{}^{\mathrm{?11}}\text{M}$ and fully saturated at $\text{1.7 · 10}{}^{\mathrm{?10}}\text{M}$ insulin. Membranes prepared from obese hyperglycemic (ob / ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at $\text{1.2 · 10}{}^{\mathrm{?10}}\text{M}$ and fully saturated at $\text{2.0 · 10}{}^{\mathrm{?10}}\text{M}$. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37°C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1–50 μM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from ‘insulin resistant’ animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

一氧化氮对心肌缺血/再灌注损伤细胞凋亡和心功能的影响

目的:采取促进或抑制NO的方法,了解在重复可逆性心肌缺血/再灌注所致的心肌顿抑时,血液中一氧化氮(NO)的动态变化与细胞顿抑及心功能的影响.方法:新西兰兔15只,随机分为3组(n=5):对照组、在静脉内注射NO合成底物L-精氨酸为L-Arg组、静脉注射一氧化氮合酶抑制剂L-硝基-精氨酸为L-NNA组.用戊巴比妥钠静脉注射麻醉后,结扎前降支制成心肌缺血/再灌注模型,用电子自旋共振法测定血液中NO含量,同时记录左心室最大上升速率dp/dtmax.将兔心肌缺血10 min,共3次,第1、2次缺血后再灌注10 min,第3次缺血后再灌注120 min.结果:第1次缺血/再灌注5 min时NO升高的顺序依次为L-Arg组最大、对照组次之,而L-NNA组较缺血前降低.而dp/dtmax明显下降的是L-Arg组最大、对照组次之、L-NNA组最小.细胞凋亡指数:L-Arg组最大,对照组次之、L-NNA组最小.结论:再灌注早期NO的大量生成及细胞凋亡参与加重心肌顿抑的过程.     

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (Y_D) in PsbD, the D2 subunit of plant photosystem II, were 50.8?±?3.5?Å, 54.9?±?4.0?Å, 57.8?±?4.9?Å, and 58.4?±?14.1?Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of Y_D relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed Y_D coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.     

Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

Kinetic,ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells

Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50–57 M, of which 30–40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (±0.2) M, very near K_d for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound⁵⁴Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. -Methyl-d,l-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme -glutamyl-cysteine synthetase prior to specific inactivation of GS by -ethyl-d,l-methionine-S,R-sulfoximine. Even inthe presence of 2–3 mM Mg(II), with only 5–10 M Mn(II) present, approximately 20–30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.     

Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel.     

两种合成抗菌肽的结构及抗菌作用机理

为深入了解两种新型人工抗菌肽mytilin-derived-peptide-1 (MDP-1) 和mytilin-derived-peptide-2 (MDP-2) 的溶液结构和抗菌机理并探讨两种抗菌肽之间活性差异的结构基础,采用二维核磁共振技术 (2-D NMR) 研究MDP分子的溶液结构;采用透射电镜技术 (Transmitted electron microscopy,TEM) 研究MDP分子对于大肠杆菌和藤黄叠球菌的作用机理。研究结果表明,MDP-1和MDP-2均采取了典型的β-发夹结构,其分子表面具有明显的疏水斑片,其分子中碱性氨基酸突出于分子表面;经MDP分子处理后的大肠杆菌以及藤黄叠球菌均出现细胞壁或细胞膜结构被破坏,并出现膜壁分离以及细胞质内缩现象。我们认为,MDP-1和MDP-2分子中的碱性氨基酸有助于MDP结合细菌表面的带负电荷的基团,同时其分子表面的疏水斑片有助于其插入到细菌细胞膜内;其疏水斑片面积以及碱性氨基酸在分子表面的拓扑结构差异是MDP-1和MDP-2活性差异的主要原因;电镜实验结果表明MDP-1和MDP-2的主要靶标是细菌细胞壁以及细胞膜;上述研究为深入了解MDP分子的结构与功能的关系以及将来基于MDP分子的药物研发奠定了基础。