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Differential Scanning Calorimetry for bacterial membranes

Differential Scanning Calorimetry for bacterial membranes



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I would like to study the freezing and melting of bacterial membranes and would like to use Differential Scanning Calorimetry (DSC) to obtain the glass transition temperature of the membrane. However, I am unsure where to find such protocols describing the process. Could someone please tell me how to use DSC for bacteria such as E. coli?


Interaction of Synthetic Polymers with Cell Membranes and Model Membrane Systems I. Differential Scanning Calorimetry

Tirrell, David A. and Boyd, Patricia M. (1981) Interaction of Synthetic Polymers with Cell Membranes and Model Membrane Systems I. Differential Scanning Calorimetry. Makromolecular Chemistry, Rapid Communications, 2 (2). pp. 193-198. ISSN 0250-9733. https://resolver.caltech.edu/CaltechAUTHORS:20150209-164526691

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Differential Scanning Calorimetry of Protein-Lipid Interactions

Differential scanning calorimetry (DSC) is a highly sensitive nonperturbing technique used for studying the thermodynamic properties of thermally induced transitions. Since these properties might be affected by ligand binding, DSC is particularly useful for the characterization of protein interactions with biomimetic membranes. The advantages of this technique over other methods consist in the direct measurement of intrinsic thermal properties of the samples, requiring no chemical modifications or extrinsic probes. This chapter describes the basic theory of DSC and provides the reader with an understanding of the capabilities of DSC instrumentation and the type of information that can be achieved from DSC studies of lipid-protein interactions. In particular, the chapter provides a detailed analysis of DSC data to assess the effects of proteins on biomimetic membranes.

Keywords: Data analysis Differential scanning calorimetry Gel to liquid–crystalline phase transition Lamellar to inverted hexagonal phase transition Lipids Lipid–protein interaction Proteins.


Langmuir monolayers and Differential Scanning Calorimetry for the study of the interactions between camptothecin drugs and biomembrane models

CPT-11 and SN-38 are camptothecins with strong antitumor activity. Nevertheless, their severe side effects and the chemical instability of their lactone ring have questioned the usual forms for its administration and have focused the current research on the development of new suitable pharmaceutical formulations. This work presents a biophysical study of the interfacial interactions of CPT-11 and SN-38 with membrane mimetic models by using monolayer techniques and Differential Scanning Calorimetry. The aim is to get new insights for the understanding of the bilayer mechanics after drug incorporation and to optimize the design of drug delivery systems based on the formation of stable bilayer structures. Moreover, from our knowledge, the molecular interactions between camptothecins and phospholipids have not been investigated in detail, despite their importance in the context of drug action. The results show that neither CPT-11 nor SN-38 disturbs the structure of the complex liposome bilayers, despite their different solubility, that CPT-11, positively charged in its piperidine group, interacts electrostatically with DOPS, making stable the incorporation of a high percentage of CPT-11 into liposomes and that SN-38 establishes weak repulsive interactions with lipid molecules that modify the compressibility of the bilayer without affecting significantly neither the lipid collapse pressure nor the miscibility pattern of drug-lipid mixed monolayers. The suitability of a binary and a ternary lipid mixture for encapsulating SN-38 and CPT-11, respectively, has been demonstrated.

Keywords: Bilayer systems Biomembrane models Camptothecins Langmuir monolayers Liposomes Membrane interactions.


Abstract

We have carried out a comparative study of the effect of cholesterol on the thermotropic phase behavior of the distearoyl and dielaidoyl molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine using high-sensitivity differential scanning calorimetry. For both molecular species of phosphatidylcholine, cholesterol incorporation produces bimodal endotherms at lower and unimodal endotherms at higher sterol concentrations. In both cases, heating and cooling endotherms are identical, and high concentrations of cholesterol (50 mol %) completely abolish the gel to liquid-crystalline phase transition. For the distearoyl molecular species of phosphatidylserine and phosphatidylethanolamine, heating and cooling endotherms are not identical, and cholesterol exhibits a considerably reduced miscibility in the gel as compared to the liquid-crystalline phase, particularly in the latter case. Thus, in neither case does the addition of 50 mol % cholesterol completely abolish the cooperative hydrocarbon chain-melting phase transition. However, the dielaidoyl molecular species of phosphatidylserine and phosphatidylethanolamine exhibit much closer correspondence in the heating and cooling modes than do the distearoyl species, and 50 mol % cholesterol is sufficient to almost or completely abolish the gel to liquid-crystalline phase transition of dielaidoylphosphatidylethanolamine and dielaidoylphosphatidylserine. In general, there is an inverse correlation between the strength of intermolecular phospholipid−phospholipid interactions, as manifested by the relative gel to liquid-crystalline phase transition temperatures of the pure phospholipids, and the miscibility of cholesterol in bilayers, particularly gel-state bilayers, formed from these phospholipids. These results indicate that the nature of cholesterol−phospholipid interactions, and thus the miscibility of cholesterol in the bilayer, depends on both the structure of the phospholipid polar headgroup and the hydrocarbon chains, as well as on the temperature and phase state of the phospholipid bilayer.


Abstract

α-Defensins are antimicrobial peptides with 29−35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded β-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil α-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.

This research was supported by grants from the Jubiläumsfonds der Österreichischen Nationalbank (Project 5100 to K.L.) and from the NIH (HL-46809 to T.G. and AI 22839 to R.I.L.).

Part of this work was published in abstract form (Lohner et al., 1995).

Correspondence should be addressed to this author at the Institut für Biophysik und Röntgenstrukturforschung, Österreichische Akademie der Wissenschaften, Steyrergasse 17/VI, A-8010 Graz, Austria. Telephone: **43-316-812004-18. Fax: **43-316-812367. Email: [email protected]


Some Applications of Calorimetry in Biochemistry and Biology

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific . Read More

Supplemental Materials

Supplemental Videos 1 and 2 Read More

Figure 1: CRISPR–Cas9-mediated DNA interference in bacterial adaptive immunity. (a) A typical CRISPR locus in a type II CRISPR–Cas system comprises an array of repetitive sequences (repeats, brown dia.

Figure 2: The mechanism of CRISPR–Cas9–mediated genome engineering. The synthetic sgRNA or crRNA–tracrRNA structure directs a Cas9 endonuclease to almost arbitrary DNA sequence in the genome through a.

Figure 3: Overall structure of Streptococcus pyogenes Cas9 (SpyCas9) in the apo state. (a) Ribbon representation of the crystal structure of SpyCas9 (PDB ID 4CMP). Individual Cas9 domains are colored .

Figure 4: Guide RNA loading enables Cas9 to form a DNA recognition–competent conformation for target search. (a) Ribbon diagram showing the apo structure of SpyCas9 aligned in the same orientation as .

Figure 5: Structures of CRISPR–Cas9 bound to DNA substrates, showing the same view as in Figure 4c after superposition. (a) Crystal structure of SpyCas9 (surface representation) in complex with sgRNA .

Figure 6: Schematic representations of the proposed mechanisms of CRISPR–Cas9-mediated target DNA recognition and cleavage. Upon sgRNA loading, Cas9 undergoes a large conformational rearrangement to r.

Figure 7: Structures of Cas9 orthologs reveal both the conserved and divergent structural features among orthologous CRISPR–Cas9 systems. Individual Cas9 domains are colored according to the scheme in.


Résumé

La calorimétrie de balayage différentielle (DSC) a été appliqué à l'étude de la stabilité et du comportement des lipopolyosides (LPS) de la membrane externe des bactéries Gram-négatives et de leur portion lipidique.

Les courbes DSC des LPS présentent des caractéristiques thermiques entre 200 et 129°C (dépolymérisation) et entre − 13 et − 36°C (transition de phase vitreuse). Ces deux aspects ont été mis en relation avec la force relative des types de liaison dans la structure de la chaîne O et avec la capacité de lier l'hydrogène intermoléculaire.

Les courbes DSC des lipides A montrent des pics endothermiques entre 40 et 24°C, autour de 15°C, et entre − 23 et − 4°C. A partir de ces résultats il a été possible d'observer de grandes différences dans le comportement thermique entre Brucella et Vibrio cholerae d'un côté, et entre Escherichia coli et Shigella flexneri d'un autre côté. La fluidité des chaînes acyles et le lyotropisme. qui sont les paramètres importants en ce qui concerne lɾxpression des activités biologiques, sont discutés à lɺide des données antérieures. Pour expliquer quelques propriétés, la fluidité peut être mise en rapport avec la température de transition de phase (βα) gel ↔ liquide critallin, qui a lieu à température physiologique. Nèanmoins, la fluidité peut être mise en rapport avec la température des caractéristiques thermiques (entre 6 et 20°C), pour lesquelles une fusion partielle de l'échantillon a été mise évidence. L�t observé entre −23° et −4°C indique lɾxistence d'une réduction forte de la concentration d⟪u du lipide A de Brucella, ce qui expliquerait le processus de fusion précoce et l➬tivité lipidique dépendante des interactions hydrophobes.


Fluidity of the Membrane (With Diagram)| Cell Biology

With the help of the technique of Electron Spin Resonance (ESR) spectroscopy, the fluidity of the membrane has been established. Harden MeConnell and O Hayes Griffith made an ex­periment using ESR spectrum after labelling the fatty acid tail of lipid layer of the membrane with a nitroxide group, having an unpaired electron.

The presence of this spin-label, i.e., nitroxide group emits energy when exposed to an external magnetic field of suitable intensity. The ESR spectrum is then noted taking a phos­pholipid compound with spin-label as control.

The spectrum of biological membrane is found to be intermediate between completely mobile and immobilised molecule (Fig. 2.9). Thus, the ESR spectra showed that lipid molecules of the biological membranes are neither in a fixed state as in crystal, nor like completely mobile molecule (fluid).

Hence the intermediate state of the membrane is referred to as liquid crystal state. The lipid molecules can move laterally within the bilayer keeping the orientation intact (i.e., hydrophilic head groups pointed towards the membrane surface and hydrophobic tails towards the membrane interior).

It is known that during the transition of one physical state to another—i.e., from solid to liquid or liquid to gas—heat is generated. Hence, the transition of the physical state in the biological membrane (i.e., from solid to liquid) has been observed by Differential Scanning Calorimetry (Fig. 2.10).

It has been observed that at low temperatures, lipid lay­ers are changed to solid or gel state .and at higher temperatures it melts to fluid or liquid crystalline state. The transition temperature, at which the change of state occurs, is below the temperature at which most physio­logical functions occur.

There are different factors which control this membrane fluidity. The increase in membrane fluidity is related with the increase of unsaturated fatty acids and decrease of fatty acid chain length and cholesterol content.

Again, the increase in membrane fluidity is inversely proportional to the transition temperature. This alteration in the physical state of the membrane has an important role in the function of the membrane. The mobility of the membrane proteins has also been established using fluorescein-labelled antibodies. The membrane proteins are capable of rapid lateral migration in fluid lipid layer.

Thus, the random distribution of membrane proteins has been observed and the behaviour of membrane proteins depends on the membrane fluidity. Thus, this model offers a dynamic picture of the membrane.


DSC is a thermoanalytical technique used to monitor heat effects, especially in the studies of polymers, liquid crystals, oxidative stability, safety screening, drug analysis and general chemical analysis. With DSC, we can analyze the heat capacity and physical transformation (e.g., glass transition, crystallization and melting) of a protein sample. The applications of DSC also include determining melting point, percent of crystallinity and crystallization kinetics, etc. Creative Biostructure provides highly sensitive and comprehensive MagHelix™ DSC service for the above mentioned purposes.


Figure. Experimental setup for a DSC experiment. (J Biomol Tech. 2010)

Calorimetry is a key technique connecting temperature and specific physical properties of substances to directly determine the enthalpy associated with the process of interest. There are various types of calorimeters, among which DSC is a thermal analysis instrument to measure how physical properties of a sample change. It is commonly used to study biochemical reactions. Just as shown in the figure, there are two types of DSC operating with different mechanisms: power-compensated DSC and heat-flux DSC. In a power-compensated DSC, the sample material is enclosed in a pan and there is another empty reference pan, the both pans are surrounded by separate furnaces and heated by separate heaters. During heating, the heater need give different power to keep the sample and empty reference at same temperature. Then the difference of thermal power that is needed to keep them at same temperature is measured. Compared with power-compensated DSC, in the instrument of heat-flux DSC such as IR (infrared)-heated DSC and SRDSC (self-reference DSC) technique, there is only one heater. The empty reference pan and the sample material pan are put on a thermoelectric disk which is surrounded by a furnace heated by a heater. Finally, the temperature difference between the reference and sample pans would be measured by area thermocouples owing to the heat capacity (Cp) of the sample. According to the thermal equivalent of Ohm's law, q=ΔT/R, the heat flow is determined. DSC is a powerful technique in the study of biochemical reactions. It facilitates to know about the thermoanalytical parameters of biomolecules. Now several new DSC heat flow measurement technique has been also developed.

Our MagHelix™ analytical methods for biophysical characterization of biomolecules include but are not limited to:

Pooria Gill, et al. Differential scanning calorimetry techniques: applications in biology and nanoscience. Journal of Biomolecular Techniques. 2010 Dec 21 (4): 167-93.


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