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CAMP-Membrane Interactions Using Fluorescence Spectroscopy
采用荧光光谱法检测环磷酸腺苷与膜的相互作用   

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Abstract

The molecular mechanism by which peptide antibiotics (also referred as cationic antimicrobial peptides-CAMPs) penetrate through the bacterial wall barrier, interact with, and disrupt their membrane is complex. It depends mainly on the peptide properties (structure, length, charge and hydrophobicity), on the characteristics of the cell wall matrix and the membrane itself.
Here, we present two fluorescence spectroscopic techniques, one for tracking the interaction of CAMPs with membranes, and the other for evaluating the ability of a peptide to cross the bacterial cell-wall and reach the membrane. The fluorescence approach is relatively simple, highly sensitive, non-invasive and allows time-scale investigation. It can be applied to lipid vesicles or intact bacteria. For membrane model systems such as liposomes, it allows to determine the binding kinetics of a peptide to vesicle and to assess the depth of penetration. By using bacterial strains carrying different mutations in their cell wall components, but not in their membrane, we can investigate how a specific element may affect the cell wall permeability to CAMPs (Saar-Dover et al., 2012).
In order to track the peptide-membrane interaction we conjugate a lipid environmentally sensitive NBD (7-nitrobenz-2-oxa-1, 3-diazole-4-yl) fluorophore to peptides. NBD fluorescence can increase up to approximately 10-fold upon interaction with membranes. Its high excitation wavelength (467 nm) and the high quantum yield reduce significantly the contribution of light scattering. NBD-labeled peptides exhibit fluorescence emission maxima around 540 nm in hydrophilic solution (Shai, 1999). However, upon interaction with lipid component such as the bacterial membrane, relocation of the NBD group into a more hydrophobic environment results in an increase in its fluorescence intensity and a blue shift of the emission maxima (Chattopadhyay and London, 1987). The first property is used to determine the binding constant of the peptide to the membrane. The second property is exploited to evaluate the depth of penetration (Merklinger et al., 2012; Zhao and Kinnunen, 2002). Here, we will focus on how to determine the binding constant. The advantage of the NBD moiety conjugation is that it allows the use of experimental conditions in which the lipid: peptide molar ratio range from < 100:1 up to > 15,000:1. The addition of NBD does not change the biological function of most of the peptide, as was found for different antimicrobial peptides such as paradaxin (Rapaport and Shai, 1992), dermaseptins (Pouny et al., 1992), cecropins (Gazit et al., 1994) and cathelicidin LL-37 (Oren et al., 1999). However, pre-examination must be done for each newly investigated peptide.

Keywords: Peptide–membrane interaction(肽-膜相互作用), Fluorescence spectroscopy(荧光光谱), Antimicrobial peptide(抗菌肽), Liposomes(脂质体), Cell wall penetration(细胞壁的渗透)

Materials and Reagents

  1. Peptides were synthesized by an Fmoc solid-phase method (Merrifield et al., 1982) on Rink amide-4-methylbenzhydrylamine hydrochloride salt (MBHA) resin. Fluorescent labeling with 4-chloro-7-nitrobenz-2-oxa-1, 3-diazole fluoride (NBD-F) or 5-(and-6)-carboxytetra-methylrhodamine succinimidyl ester (Rhodamine) was followed by peptide cleavage from the resin and purification by reverse phase high-performance liquid chromatography (RP-HPLC). See detailed methods in Oren et al. (1999) and Avrahami et al. (2001).
  2. Liposome suspension stock at total lipid concentration of 12.5 mM. For liposome preparation see Kliger et al. (1997)
  3. Bacterial suspension (OD600 nm adjusted to 4)
  4. Fluorescently labeled peptides solution (see Note 1)
  5. Double Distilled water (DDW) or Milli-Q reagent grade water
  6. Phosphate buffered saline (PBS) (pH 7.4)
  7. 70% (v/v) ethanol
  8. 5 x 5 mm quartz cuvette

Equipment

  1. Automatic peptide synthesizer ABI 433A (Applied Biosystems)
  2. Reverse phase high-performance liquid chromatography (RP-HPLC) Agilent HPLC 1100 (Hewlett Packard)
  3. SLM-Aminco Bowman series 2-luminescence spectrophotometer FA-355 (SLM-Aminco)

Procedure

  1. Binding of NBD-labeled peptide to membranes
    The binding constant of a peptide is calculated from a titration of lipid vesicles, either small-unilamellar vesicles (SUVs, 10-50 μm size) or large-unilamellar vesicles (LUVs, 50-100 μm size) into NBD-peptide solution. To achieve an accurate result, at least 20 points should be recorded for each curve. An accepted dilution factor of the peptide solution is up to 10% and therefore no more than 40 μl of vesicle solution should be added. All assays were performed in room temperature (22-24 °C).
    1. Dissolve NBD-peptide of the requested solution (such as DDW or PBS) to a final concentration of 0.1 μM (see Note 2). Each measurement uses 400 μl of peptide solution, therefore prepare 810 μl for two repetitions.
    2. Set the spectrophotometer to spectrum mode with excitation wavelength of 467 nm and emission wavelength of 500-600 nm. In our device, slits are usually set to 5-10 nm. Using wider slits will improve sensitivity but can increase background noise and therefore should be individually determined for each peptide.
    3. Add 400 μl peptide solution to a pre-cleaned cuvette (magnetic stirrer can be used) and read the signal output. Measure again every few minutes until no change is detected. This is the basal signal of the labeled peptide in the absence of membrane compounds.
    4. Add 1 μl from the LUVs suspension stock to the cuvette to reach an initial peptide/lipid ratio of 1:312 and read again. Calculation of ratio: 12.5 mM lipid is 12,500 μM that are being diluted 1:400 in a solution containing 0.1 μM peptide. Therefore, when 1 μl are added the ratio is 1:312 (12,500/401*10). When another 1 μl will be added the ratio will be 1: 622 and so on.
    5. Re-measure the signal intensity every 1 min until no change in the signal is detected. This will indicate that binding has reached equilibrium.
    6. Repeat step 1-d successively until no change in the peak maxima (around 530 nm) can be detected.
    7. Clean the cuvette by washing it three times with 70% (v/v) ethanol. Trace ethanol in the cuvette should be removed by rinsing with DDW.
    8. To account for background, the emissions of the vesicles alone at the same wavelength should be monitored and subtracted. Therefore, repeat steps 1-c~f using the same solvents but without dissolving peptide in it.

  2. Cell-wall permeability assay
    The assay is designed to compare the ability of a given peptide to penetrate the cell wall of a given bacterial strain and interact with its membrane (Saar-Dover et al., 2012). The relative elevation in NBD emission should be calculated for each strain and compared.
    1. Grow you bacteria to an exponential stage, concentrate cells from the culture by centrifuging 5 ml at 1,300 x g), 3 min. Wash and re-centrifuge pallet twice with PBS. Adjust your bacterial suspension to OD600 nm = 4 in PBS.
    2. Dissolve NBD-peptide in PBS solution to a final concentration of 0.1-1 μM (use concentration that does not disrupt the cellular integrity, this can be determined separately using a SYTOX green assay (Saar-Dover et al., 2012).
    3. Set the spectrophotometer to kinetic mode with excitation wavelength of 467 nm and emission wavelength of 530 nm. In our device, SLM-Aminco Bowman series 2-luminescence spectrophotometer, slits are usually set to 5-10 nm. Using wider slits will improve sensitivity but can increase background noise and therefore should be individually determined for each peptide.
    4. Add 400 μl peptide solution to a pre-cleaned cuvette (magnetic stirrer can be used) and read the signal output until it stabilizes. This is the basal signal of the labeled peptide in the absence of membrane compounds.
    5. Add 10 μl from the bacterial suspension to the cuvette. Track the change in signal intensity with time until equilibrium is reached.
    6. Clean the cuvette by washing it three times with 70% (v/v) ethanol. Trace ethanol in the cuvette should be removed by rinsing with DDW.
    7. To account for background, the emissions of bacteria alone at the same wavelength should be monitored and subtracted. Therefore, repeat steps 2-c~e using the same solvents but without dissolving peptide in it.
    8. The signal intensity can be affected by oligomerization of the labeled peptide over the bacterial surface and self-quenching (reduced intensity). We therefore assess the level of peptide oligomerization by repeating the experiment using Rhodamine labeled peptides. Rhodamine is highly sensitive to quenching but unlike NBD, its emission is not affected strongly by the polarity of its environment.

Calculations


     For binding of NBD-labeled peptide to membranes:

  1. Prepare a table of titration results- emission (Y) versus lipid concentration (X).
  2. Subtract the baseline value (solution only) from each Y value to correct for background.
  3. You should get a saturation curve, meaning a non-linear curve (Figure 1). Use a non-linear equation program solver (such as GraphPad Prism) to extract the best fitted equation.


Figure 1. Binding of NBD-labeled peptide to membranes. A representative saturation curve describing an increase in NBD fluorescence upon titration of phosphatidylcholine: cholesterol (9:1) large unilamellar lipid vesicles (LUVs) into 0.2 μM NBD conjugated peptide (NBD-gp41 TMD). Nonlinear least-squares analysis was used to determine the affinity constant (Ka).


  1. Calculate the Ka (association constant). You can also determine the peptide: lipid ratio at saturation.
  2. Alternatively, a less preferable but still applicable way will be to calculate the slope from the linear part of you curve, as long as there are at least 10 points in that region.
  3. See more details in Rosenfeld et al. (2006).


     For cell-wall permeability assay:

  1. Subtract the bacterial basal emission from the final emission value recorded after bacteria were added to peptide and the signal has stabilized.
  2. Calculate (in percentage) how much the addition of bacteria increased the emission relatively to the basal peptide emission.
  3. Compare the degree of change between any given bacterial strains (wild type versus mutants for example) to evaluate the role of a given component to cell-wall permeability to peptides.
  4. See more details in Saar-Dover et al. (2012).

Notes

  1. Peptide concentration (C) in molar units is determined spectroscopic using the Beer–Lambert equation:

    Where A is the actual absorbance at 467 nm (NBD) and at 530 nm (rhodamine). The molar absorption coefficient (ε) of NBD is 16,000 [cm/M], and that of rhodamine is 38,000 [cm/M]. L is the cuvette path length in centimeter.
  2. Peptide concentration for the experiment should be at the low micromolar rang to reach very low peptide/lipid ratio and is also dependent on the labeling and purification quality. We generally use a concentration range of 0.1-1 μM of labeled peptides.

References

  1. Avrahami, D., Oren, Z. and Shai, Y. (2001). Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides. Biochemistry 40(42): 12591-12603.
  2. Chattopadhyay, A. and London, E. (1987). Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26(1): 39-45. 
  3. Gazit, E., Lee, W. J., Brey, P. T. and Shai, Y. (1994). Mode of action of the antibacterial cecropin B2: a spectrofluorometric study. Biochemistry 33(35): 10681-10692.
  4. Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. and Shai, Y. (1997). Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell Fusion. Structure-function study. J Biol Chem 272(21): 13496-13505. 
  5. Merklinger, E., Gofman, Y., Kedrov, A., Driessen, A. J., Ben-Tal, N., Shai, Y. and Rapaport, D. (2012). Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors. Biochem J 442(2): 381-389.
  6. Merrifield, R. B., Vizioli, L. D. and Boman, H. G. (1982). Synthesis of the antibacterial peptide cecropin A (1-33). Biochemistry 21(20): 5020-5031.
  7. Oren, Z., Lerman, J. C., Gudmundsson, G. H., Agerberth, B. and Shai, Y. (1999). Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 341 ( Pt 3): 501-513.
  8. Pouny, Y., Rapaport, D., Mor, A., Nicolas, P. and Shai, Y. (1992). Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 31(49): 12416-12423.
  9. Rapaport, D. and Shai, Y. (1992). Aggregation and organization of pardaxin in phospholipid membranes. A fluorescence energy transfer study. J Biol Chem 267(10): 6502-6509.
  10. Rosenfeld, Y., Papo, N. and Shai, Y. (2006). Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J Biol Chem 281(3): 1636-1643. 
  11. Saar-Dover, R., Bitler, A., Nezer, R., Shmuel-Galia, L., Firon, A., Shimoni, E., Trieu-Cuot, P. and Shai, Y. (2012). D-alanylation of lipoteichoic acids confers resistance to cationic peptides in group B Streptococcus by increasing the cell wall density. PLoS Pathog 8(9): e1002891.
  12. Shai, Y. (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462(1-2): 55-70. 
  13. Zhao, H. and Kinnunen, P. K. (2002). Binding of the antimicrobial peptide temporin L to liposomes assessed by Trp fluorescence. J Biol Chem 277(28): 25170-25177.

简介

肽抗生素(也称为阳离子抗微生物肽-CAMP)穿透细菌壁屏障,与其相互作用并破坏其膜的分子机制是复杂的。它主要取决于肽的性质(结构,长度,电荷和疏水性),细胞壁基质的特性和膜本身。
在这里,我们提出两种荧光光谱技术,一种用于跟踪CAMP与膜的相互作用,另一种用于评估肽穿过细菌细胞壁并到达膜的能力。荧光方法相对简单,高度敏感,非侵入性,并允许时间尺度的调查。它可以应用于脂质囊泡或完整细菌。对于膜模型系统例如脂质体,其允许确定肽与囊泡的结合动力学并且评估渗透的深度。通过使用在其细胞壁组分中携带不同突变但在其膜中不具有不同突变的细菌菌株,我们可以研究特定元件如何影响CAMP的细胞壁通透性(Saar-Dover等人,2012 )。
为了跟踪肽 - 膜相互作用,我们将脂质环境敏感的NBD(7-硝基苯并-2-氧杂-1,3-二唑-4-基)荧光团与肽缀合。 NBD荧光在与膜相互作用时可以增加高达约10倍。其高激发波长(467nm)和高量子产率显着降低了光散射的贡献。 NBD标记的肽在亲水溶液中表现出约540nm的荧光发射最大值(Shai,1999)。然而,当与脂质组分例如细菌膜相互作用时,NBD基团重新定位到更疏水的环境导致其荧光强度的增加和发射最大值的蓝移(Chattopadhyay和London,1987)。第一性质用于确定肽与膜的结合常数。第二个属性被用于评估渗透的深度(Merklinger等人,2012; Zhao和Kinnunen,2002)。在这里,我们将关注如何确定结合常数。 NBD部分缀合的优点是其允许使用实验条件,其中脂质:肽摩尔比范围从<100:1直至> 15,000:1。添加NBD不改变大多数肽的生物学功能,如对于不同的抗微生物肽例如paradaxin(Rapaport和Shai,1992),dermaseptins(Pouny等人,1992)所发现的, ,杀菌肽(Gazit等人,1994)和cathelicidin LL-37(Oren等人,1999)。但是,必须对每个新研究的肽进行预检查

关键字:肽-膜相互作用, 荧光光谱, 抗菌肽, 脂质体, 细胞壁的渗透

材料和试剂

  1. 在Rink酰胺-4-甲基二苯甲基胺盐酸盐(MBHA)树脂上通过Fmoc固相法(Merrifield等人,1982)合成肽。 用4-氯-7-硝基苯并-2-氧杂-1,3-二唑氟化物(NBD-F)或5-(和-6) - 羧基四甲基罗丹明琥珀酰亚胺酯(罗丹明)进行荧光标记,然后从 树脂并通过反相高效液相色谱(RP-HPLC)纯化。 请参阅Oren等人中的详细方法。 (1999)和Avrahami等人。 (2001)。
  2. 脂质体悬浮液母液,总脂质浓度为12.5mM。 对于脂质体制备,参见Kliger等人。 (1997)
  3. 细菌悬浮液(OD <600nm,调节至4)
  4. 荧光标记肽溶液(见注1)
  5. 双蒸馏水(DDW)或Milli-Q试剂级水
  6. 磷酸盐缓冲盐水(PBS)(pH 7.4)
  7. 70%(v/v)乙醇
  8. 5 x 5 mm石英比色皿

设备

  1. 自动肽合成仪ABI 433A(Applied Biosystems)
  2. 反相高效液相色谱(RP-HPLC)Agilent HPLC 1100(Hewlett Packard)
  3. SLM-Aminco Bowman系列2-发光分光光度计FA-355(SLM-Aminco)

程序

  1. NBD标记肽与膜的结合
    肽的结合常数由脂质囊泡(小单层囊泡(SUV,10-50μm大小)或大单层囊泡(LUV,50-100μm大小)滴定到NBD-肽溶液中计算)。为了获得准确的结果,应为每条曲线记录至少20个点。肽溶液的接受稀释倍数高达10%,因此应当加入不超过40μl的囊泡溶液。所有测定在室温(22-24℃)下进行
    1. 将所需溶液(例如DDW或PBS)的NBD肽溶解至终浓度为0.1μM(见注2)。每次测量使用400μl肽溶液,因此准备两个810μl 重复。
    2. 将分光光度计设置为具有467nm的激发波长和500-600nm的发射波长的光谱模式。在我们的设备中,狭缝通常设置为5-10 nm。使用更宽的狭缝将改善灵敏度,但是可以增加背景噪声,因此应该对每个肽单独确定。
    3. 加入400μl肽溶液到预先清洗的比色皿(磁力搅拌器可以使用),并读取信号输出。每隔几分钟再次测量,直到没有检测到变化。这是在没有膜化合物的情况下标记肽的基础信号
    4. 从LUVs悬浮液添加1微升到比色杯,以达到1:312的初始肽/脂质比,并再次读取。计算比率:12.5mM脂质是12,500μM,其在含有0.1μM肽的溶液中以1:400稀释。因此,当加入1μl时,比例为1:312(12,500/401×10)。当另外1μl将被添加的比例将是1:622等等。
    5. 每1分钟重新测量信号强度,直到没有检测到信号的变化。这将表明装订已达到平衡。
    6. 重复步骤1-d,直到峰最大值(约530nm)没有变化可以检测到
    7. 通过用70%(v/v)乙醇洗涤三次来清洁比色杯。比色杯中的痕量乙醇应通过用DDW冲洗除去。
    8. 为了考虑背景,应该监测和扣除在相同波长下单独的囊泡的发射。因此,使用相同的溶剂,但不溶解肽,重复步骤1-c〜f
  2. 细胞壁渗透性测定
    设计该测定法以比较给定肽穿透给定细菌菌株的细胞壁并与其膜相互作用的能力(Saar-Dover等人,2012)。应该计算每个应变的NBD发射的相对高度并进行比较。
    1. 生长你的细菌到指数阶段,通过离心5毫升在1,300×g ,从培养浓缩细胞,3分钟。洗涤并用PBS再次离心平板两次。在PBS中将细菌悬浮液调节至OD <600> nm = 4。
    2. 将NBD肽溶解在PBS溶液中至终浓度为0.1-1μM(使用不破坏细胞完整性的浓度,这可以使用SYTOX绿色测定法(Saar-Dover等人) 。,2012)。
    3. 将分光光度计设置为激发波长为467nm和发射波长为530nm的动力学模式。在我们的设备中,SLM-Aminco Bowman系列2-发光分光光度计,狭缝通常设置为5-10nm。使用更宽的狭缝将改善灵敏度,但是可以增加背景噪声,因此应该对每个肽单独确定。
    4. 加入400μl肽溶液到预先清洗的比色皿(磁力搅拌器可以使用),读取信号输出,直到它稳定。这是在没有膜化合物的情况下标记肽的基础信号
    5. 加入10微升从细菌悬浮液到比色杯。跟踪信号强度随时间的变化,直到达到平衡。
    6. 通过用70%(v/v)乙醇洗涤三次来清洁比色杯。比色杯中的痕量乙醇应通过用DDW冲洗除去。
    7. 为了考虑背景,应该监测和减去在相同波长下单独的细菌的排放。 因此,使用相同的溶剂,但不溶解肽,重复步骤2-c〜e
    8. 信号强度可受标记肽在细菌表面上的寡聚化和自淬灭(降低的强度)的影响。 因此,我们通过使用罗丹明标记的肽重复实验来评估肽寡聚化的水平。 若丹明对淬灭非常敏感,但与NBD不同,其发射不受其环境极性的强烈影响

计算


     对于NBD标记的肽与膜的结合:

  1. 准备一个滴定结果表 - 发射(Y)与脂质浓度(X)
  2. 从每个Y值中减去基线值(仅解决方案)以校正背景
  3. 您应该得到饱和曲线,意味着非线性曲线(图1)。 使用非线性方程程序解算器(如GraphPad Prism)提取最佳拟合方程。


图1. NBD标记的肽与膜的结合。 描述在磷脂酰胆碱:胆固醇(9:1)大单层脂质囊泡(LUV)滴定至0.2μMNBD缀合肽(NBD-gp41 TMD)中时NBD荧光增加的代表饱和曲线。 使用非线性最小二乘法分析来确定亲和常数(Ka)。


  1. 计算K sub(缔合常数)。 您还可以确定饱和时的肽:脂质比例。
  2. 或者,一个不太优选但仍然适用的方法是从曲线的线性部分计算斜率,只要该区域至少有10个点。
  3. 详情请参阅Rosenfeld 。 (2006)。


     对于细胞壁渗透性测定:

  1. 从细菌加入肽中并且信号稳定后记录的最终发射值减去细菌基础发射
  2. 计算(以百分比)细菌的添加相对于基础肽排放增加了多少发射
  3. 比较任何给定的细菌菌株(例如野生型与突变体)之间的变化程度以评估给定组分对细胞壁对肽的渗透性的作用。
  4. 详情请参阅Saar-Dover 等人。 (2012)。

笔记

  1. 使用Beer-Lambert方程通过光谱法测定以摩尔单位表示的肽浓度(em):</em>
    其中A em是在467nm(NBD)和在530nm(罗丹明)的实际吸光度。 NBD的摩尔吸光系数(ε)为16000 [cm/M],罗丹明的摩尔吸光系数为38000 [cm/M]。 是以厘米为单位的比色杯路径长度。
  2. 实验的肽浓度应该在低微摩尔范围以达到非常低的肽/脂质比,并且还取决于标记和纯化质量。我们通常使用0.1-1μM标记肽的浓度范围

参考文献

  1. Avrahami,D.,Oren,Z.and Shai,Y。(2001)。 多种脂肪族氨基酸取代对非对映体膜活性的结构,功能和作用模式的影响肽。生物化学 40(42):12591-12603
  2. Chattopadhyay,A。和London,E。(1987)。 视差法,利用自旋标记磷脂的荧光猝灭直接测量膜渗透深度。 Biochemistry 26(1):39-45。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Saar-Dover, R. and Shai, Y. (2013). CAMP-Membrane Interactions Using Fluorescence Spectroscopy. Bio-protocol 3(15): e846. DOI: 10.21769/BioProtoc.846.
  2. Saar-Dover, R., Bitler, A., Nezer, R., Shmuel-Galia, L., Firon, A., Shimoni, E., Trieu-Cuot, P. and Shai, Y. (2012). D-alanylation of lipoteichoic acids confers resistance to cationic peptides in group B Streptococcus by increasing the cell wall density. PLoS Pathog 8(9): e1002891.
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