Fazale R. Rana

Interactions between the antimicrobial peptide, magainin 2, and Salmonella typhimurium lipopolysaccharides.

Using FT‐IR spectroscopy, the effects of magainin 2 on the thermotropic behavior of LPS isolated from wild‐type (SL3770) and LPS‐mutant strains of Salmonella typhimurium are characterized and compared. The mutant strains include Ra (SL3749), polymyxin‐sensitive Rb2(s) (SH5014), polymyxin‐resistant Rb2(r) (SH5357) and Rc (HN202) LPS chemotypes, whose polysaccharide chains differ in length but possess an identical number of phosphorylation sites. In all cases, magainin 2 causes a concentration‐dependent disordering of the LPS fatty acyl chains. Differences in disordering of LPS correlate more closely with the charge on the LPS molecule (determined by high‐resolution 31P NMR) rather than with the length of the LPS sugar side chain, contradicting the currently accepted model for the interaction of cationic antibiotics with the Gram‐negative cell envelope.

Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT‐IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature‐dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is −7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram‐negative cell envelope and may act as a molecular sponge to protect the plasma membrane.

An Automated Temperature Control and Data Acquisition System for Fourier Transform Infrared Studies: Application to Phase Changes in Hydrocarbons and Lipid Bilayers

As a means of facilitating temperature-dependent FT-IR studies of liquid samples, a simple, accurate, efficient, and versatile system for automated thermoelectric temperature control and UNIX-based data acquisition is described. With the use of this system, phase changes in an n-alkane, pentadecane, and an aqueous dispersion of a phospholipid, dipalmitoylphosphatidylcholine, were characterized. The combination of precise temperature regulation, ability to quantitate very small changes in the infrared bands, and careful control of the thermal history of the sample reveals subtle changes in the physical structure of these molecules in a reproducible manner.

Combined Infrared and 31P NMR Spectroscopic Method for Determining the Fractional Composition in Langmuir-Blodgett Films of Binary Phospholipid Mixtures

A method has been developed to determine the exact fractional composition of binary mixtures of phospholipids at the air/water (A/W) interface by infrared spectroscopy in combination with 31P NMR spectroscopy and Langmuir-Blodgett surface chemistry. This procedure utilizes the wavenumber shift observed upon the synthetic replacement of hydrogen with deuterium in the lipid acyl chains to separate the vibrational bands due to each component in mixtures of deuterated and normally proteated lipids. Classical Langmuir-Blodgett monolayer transfer techniques are used to transfer the binary lipid mixture from the A/W interface onto Ge crystals, where their IR spectra may be obtained with the use of attenuated total reflectance (ATR) infrared spectroscopy. The ratio of the integrated areas for the symmetric C-H and C-D stretching vibrations in the IR spectrum of the transferred monolayer film is used to determine fractional composition empirically; these vibrational intensities can be related to fractional composition in the following way: First, the integrated area ratios of the symmetric C-H and C-D stretching vibrations are obtained from the IR spectra of a standard series of binary mixtures of proteated and deuterated lipids at varying mole ratios. Second, these integrated area ratios obtained from the IR spectra of standards are correlated with the exact mole fraction of each phospholipid component in the mixture. The exact mole fractions are determined by the intensities of the phospholipid peaks obtained from high-resolution, solution-phase 31P NMR signals of the same set of standard mixtures that was used to prepare the IR samples. High-resolution 31P NMR spectroscopy in the presence of a line-narrowing reagent is a method that can provide an independent spectroscopic means of determining mole ratios in a binary solution of phospholipids. This IR-NMR comparison method essentially establishes a calibration curve for the IR C-D:C-H integrated intensity ratio in the binary mixture versus mole fraction as determined by NMR. Third, the IR C-D:C-H ratio in monolayer films of the binary phospholipid mixture transferred from the A/W interface to Ge crystals may be used to calculate the mole fraction of each component on the basis of the IR-NMR calibrations established in the IR spectra of the binary mixture standards. This technique was used to measure the fractional composition of binary mixtures of phospholipids at the A/W interface containing acyl chain perdeuterated DPPC (i.e., DPPC-[d62]) in combination with acyl chain proteated DPPG. The Langmuir-Blodgett technique was used to transfer monomolecular films of DPPC-[d62]: DPPG mixtures to a Ge ATR crystal at 40 mN m−1. The mole fraction of each component was estimated from the relative intensities of the symmetric C-D and C-H stretching bands by the use of ATR-infrared spectroscopy in conjunction with the line-narrowed 31P NMR spectra of the phospholipid solutions. This model system was chosen for study due to its relevance in evaluating the proposed “squeezing out” mechanism of pulmonary surfactant physiology, in which the surfactant is proposed to reduce the surface tension of the air/alveolar lining by excluding all components except DPPC from the surface layer upon compression. Using this combined infrared/31P NMR calibration method, we find no evidence for the exclusion of any surface component upon compression of this mixed film.

Determination of the lipid composition of Salmonella typhimurium outer membranes by 31P NMR

Abstract High-resolution 31 P NMR spectroscopy is used to determine the phospholipid composition and the relative amounts of lipopolysaccharide (LPS) and phospholipid in the outer membrane (OM) of wild-type Salmonella typhimurium and rfb, rfa and pmrA mutants, which produce LPS molecules with a deficient sugar side chain. LPS mutations do not alter the phospholipid composition of the outer membrane and have only a limited effect on the relative levels LPS and phospholipids. PE comprises 85–95 mol % of the OM phospholipids. The mole fraction of LPS and phospholipids in the OM is 0.3–0.4 and 0.6–0.7, respectively. LPS mutations do not influence the fatty acid composition of either LPS or OM phospholipids. These data show that 31 P NMR is a powerful tool for identifying and quantitating bacterial membrane lipids and provide a foundation for understanding the molecular interactions of these lipids with cationic antimicrobial peptides, such as defensins, magainins and polymyxin B.